A Novel Complex Comprising A Cell Penetrating Peptide, A Cargo And A TLR Peptide Agonist

ABSTRACT

The present invention provides a novel complex comprising a) a cell penetrating peptide, b) at least one antigen or antigenic epitope, and c) at least one TLR peptide agonist, wherein the components a)-c) are covalently linked. Moreover, the present invention also provides a nucleic acid encoding such a complex, wherein the complex is a peptide or a protein. Such a nucleic acid may be comprised by a vector, and such a vector may be comprised by a host cell. In particular, compositions, such as a pharmaceutical compositions and vaccines are provided, which may be useful for example in the prevention and/or treatment of a diseases and/or a disorder including cancer, hematological disorders, infectious diseases, autoimmunity disorders and transplant rejections.

The present invention relates to the field of vaccination, in particularto cancer vaccines.

The immune system can recognize and to some extent eliminate tumorcells, however, this anti-tumor response is often of low amplitude andinefficient. Boosting this weak anti-tumor response with therapeuticvaccination has been a long sought goal for cancer therapy. Modulatingthe immune system to enhance immune responses has thus become apromising therapeutic approach in oncology as it can be combined withstandard of care treatments.

Promising preclinical data and advances in clinical trials, includingthe recent FDA approval of the Sipuleucel-T vaccine and of theanti-CTLA-4 antibody, show that active immunization is a safe andfeasible treatment modality for certain cancer types. Induction oftumor-specific cytotoxic T lymphocytes (CTLs) mediated immune responseshas been reported using different approaches including modified tumorcell vaccines, peptide vaccines, recombinant viral vectors, DNA,protein, or dendritic cell vaccines. However, the anti-tumoral immunitymediated by CTLs only occasionally correlates with tumor regression andonly a few projects have reached the phase III clinical stage.

Overall, cancer vaccines showed very limited clinical efficacy so far.Indeed, at the end of 2011, amongst the 300 hundred ongoing cancervaccine clinical trials, only 19 phase III trials were reported(globaldata, 2014. Amongst them, there are NeuVax, a peptide vaccine forbreast cancer, Stimuvax, a liposome based vaccine for Non-Small CellLung Carcinoma (NSCLC) and breast cancer, TG4010, a vaccinia-basedvaccine for NSCLC and GSK1572932A, an adjuvanted liposome for NSCLC.These four cancer vaccines are based on different technologies and havein common that they are targeting one single antigen.

Therapeutic cancer vaccines can be divided into two principalcategories: personalized (autologous) and standardized vaccines, andfurther classified depending on the technology platform. Currentpersonalized vaccines include tumor lysate vaccines as well as dendriticcells based vaccine (hereinafter cell based). For the latter, antigenloading can occur either with a pulse using tumor lysates, ortransfection with RNA extracted from the tumors. In this case, theantigens are tumor specific or associated, but are not clearly defined.Dendritic cells can also be loaded with defined antigens either withpeptide pulse or using a protein such as the Prostatic Acid Phosphatase(PAP) used to engineer the Provenge® vaccine. However, the manufacturingprocess of these cell-based therapies is time-consuming andlabor-intensive while quality standards are difficult to reach andmaintain. Immunomonitoring creates further complications. Moreover, themajority of the autologous cancer vaccines do not allow the identitiesor quantities of antigens used to be controlled, unlike defined andstandardized vaccines.

In contrast to cell-based therapy (APCs, T cells, CARs, lysates),subunits vaccines (protein or peptides) allow the development of astandardized vaccine with an easier production and significantly betterbatch to batch reproducibility that can be administrated to a broadrange of patients. Furthermore, the antigens are fully defined allowingfor better immune-monitoring and reducing the risk of unwanted effectsof vaccine component.

The different approaches which were evaluated in pre-clinical andclinical development include short peptide vaccines (Slingluff C L, Jr.The present and future of peptide vaccines for cancer: single ormultiple, long or short, alone or in combination? Cancer journal 2011;17(5):343-50), long-peptide vaccines (Melief C J, van der Burg S H.Immunotherapy of established (pre)malignant disease by synthetic longpeptide vaccines. Nature reviews Cancer 2008; 8(5):351-60) and proteins.In contrast to long peptide and protein vaccines, short peptide vaccineshave a very short half-life and can have negative consequences on theimmune response.

For the protein-based vaccines, the results of targeting MAGE-A3 with arecombinant fusion protein-based vaccine have been enthusiasticallyawaited after promising phase II data in metastatic melanoma (Kruit W H,Suciu S, Dreno B, Mortier L, Robert C, Chiarion-Sileni V, et al.Selection of immunostimulant AS15 for active immunization with MAGE-A3protein: results of a randomized phase II study of the EuropeanOrganisation for Research and Treatment of Cancer Melanoma Group inMetastatic Melanoma. Journal of clinical oncology: official journal ofthe American Society of Clinical Oncology 2013; 31(19):2413-20) andnon-small cell lung cancer (NSLC) (Vansteenkiste J, Zielinski M, LinderA, Dahabreh J, Gonzalez E E, Malinowski W, et al. Adjuvant MAGE-A3immunotherapy in resected non-small-cell lung cancer: phase IIrandomized study results. Journal of clinical oncology: official journalof the American Society of Clinical Oncology 2013; 31(19):2396-403).However, in 2013 the phase III DERMA trial in melanoma (NCT00796445) didnot meet its first co-primary endpoint, followed in 2014 with a stop ofthe phase III MAGRIT study in NSCL (NCT00480025). Despite these verydisappointing clinical results, protein based vaccines undeniablypresent many advantages.

A therapeutic cancer vaccine is administrated to cancer patients tostrengthen the capability of their immune system to recognize and killthe tumor cells. The main goal of a therapeutic cancer vaccine is togenerate killer T cells (also called cytotoxic T lymphocytes) specificfor the tumor cells. To this end and to achieve a potent immuneresponse, the vaccine must contain molecules called antigens that arealso present in the tumor and that need to be delivered to AntigenPresenting Cells (APCs), especially dendritic cells (DCs), to allowcancer immunity to be initiated. The DCs process these tumor antigensinto small peptides that are presented on cell surface expressed MHCclass I or MHC class II molecules to T cells. Peptides that are thenrecognized by T cells and thereby induce their stimulation are calledepitopes. Presentation by MHC class I and MHC class II molecules allowsactivation of two classes of T cells, CD8⁺ cytotoxic T lymphocytes(CTLs) and CD4⁺ helper T (T_(h)) cells, respectively. In addition, tobecome fully activated, beside antigen recognition T cells require asecond signal, the co-stimulatory signal, which is antigen non-specificand is provided by the interaction between co-stimulatory moleculesexpressed on the surface of APCs and the T cell. Therefore two majorrequirements for an efficient therapeutic cancer vaccine are thespecificity of the tumor antigens and the ability to deliver themefficiently to DCs.

Taken together, induction of a tumor specific immune response thusrequires three main steps: (i) an antigen must be delivered to dendriticcells, which will process it into epitopes, (ii) dendritic cells shouldreceive a suitable activation signal, and (iii) activated tumorantigen-loaded dendritic cells must generate T-cell mediated immuneresponses in the lymphoid organs.

Since tumor cells can escape the immune system by down-regulatingexpression of individual antigens (passive immune escape),multi-epitopic antigen delivery provides an advantage. Indeed, proteinbased vaccines allow multi-epitopic antigen delivery to antigenpresenting cells (APCs) such as dendritic cells (DCs) without thelimitation of restriction to a single MHC allele. Another strength islong-lasting epitope presentation recently described in dendritic cellsloaded with proteins (van Montfoort N, Camps M G, Khan S, Filippov D V,Weterings J J, Griffith J M, et al. Antigen storage compartments inmature dendritic cells facilitate prolonged cytotoxic T lymphocytecross-priming capacity. Proceedings of the National Academy of Sciencesof the United States of America 2009; 106(16):6730-5). Furthermore,proteins require uptake and processing by DCs to achieve MHC restrictedpresentation of their constituent epitopes. This reduces the risk ofinducing peripheral tolerance as has been shown after vaccination withshort peptides that do not have such stringent processing requirements(Toes R E, Offringa R, Blom R J, Melief C J, Kast W M. Peptidevaccination can lead to enhanced tumor growth through specific T-celltolerance induction. Proceedings of the National Academy of Sciences ofthe United States of America 1996; 93(15):7855-60).

However, most soluble proteins are generally degraded in endolysosomesand are poorly cross-presented on MHC class I molecules and aretherefore poorly immunogenic for CD8⁺ T cell responses (Rosalia R A,Quakkelaar E D, Redeker A, Khan S, Camps M, Drijfhout J W, et al.Dendritic cells process synthetic long peptides better than wholeprotein, improving antigen presentation and T-cell activation. Europeanjournal of immunology 2013; 43(10):2554-65). Moreover, although matureDCs are more potent than immature DCs in priming and eliciting T-cellresponses (Apetoh L, Locher C, Ghiringhelli F, Kroemer G, Zitvogel L.Harnessing dendritic cells in cancer. Semin Immunol. 2011; 23:42-49),they lose the ability to efficiently take up exogenous antigens,particularly for MHC class II restricted antigens (Banchereau J,Steinman R M. Dendritic cells and the control of immunity. Nature. 1998;392:245-252). As a result, peptide-pulsed DCs as vaccines have severallimitations. For example, peptide degradation, rapid MHC class Iturnover, and the disassociation of peptide from MHC class I moleculesduring the preparation and injection of DC/peptides may result in shorthalf-lives of MHC class I/peptide complexes on the DC surface, leadingto weak T-cell responses.

To improve the efficacy of protein-based vaccine delivery, the use ofcell penetrating peptides for intracellular delivery of cancer peptidesinto DCs has been proposed (Wang R F, Wang H Y. Enhancement of antitumorimmunity by prolonging antigen presentation on dendritic cells. NatBiotechnol. 2002; 20:149-156). Cell penetrating peptides (CPPs) arepeptides of 8 to 40 residues that have the ability to cross the cellmembrane and enter into most cell types (Copolovici D M, Langel K,Eriste E, Langel U. Cell-penetrating peptides: design, synthesis, andapplications. ACS nano 2014; 8(3):1972-94, Milletti F. Cell-penetratingpeptides: classes, origin, and current landscape. Drug Discov Today2012). Alternatively, they are also called protein transduction domain(PTDs) reflecting their origin as occurring in natural proteins. Severalpotent CPPs have been identified from proteins, including the Tatprotein of human immunodeficiency virus, the VP22 protein of herpessimplex virus, and fibroblast growth factor (Berry C C. Intracellulardelivery of nanoparticles via the HIV-1 tat peptide. Nanomedicine. 2008;3:357-365; Deshayes S, Morris M C, Divita G, Heitz F. Cell-penetratingpeptides: Tools for intracellular delivery of therapeutics. Cell MolLife Sci. 2005; 62:1839-1849; Edenhofer F. Protein transductionrevisited: Novel insights into the mechanism underlying intracellulardelivery of proteins. Curr Pharm Des. 2008; 14:3628-3636; Gupta B,Levchenko T S, Torchilin V P. Intracellular delivery of large moleculesand small particles by cell-penetrating proteins and peptides. Adv DrugDeliv Rev. 2005; 57:637-651; Torchilin V P. Recent approaches tointracellular delivery of drugs and DNA and organelle targeting. AnnuRev Biomed Eng. 2006; 8:343-375). It was found that T-cell activityelicited by DC/TAT-TRP2 was 3- to 10-fold higher than that induced byDC/TRP2 (Wang H Y, Fu T, Wang G, Gang Z, Donna M P L, Yang J C, RestifoN P, Hwu P, Wang R F. Induction of CD4+ T cell-dependent antitumorimmunity by TAT-mediated tumor antigen delivery into dendritic cells. JClin Invest. 2002a; 109:1463-1470).

Moreover, subunits vaccines (peptides or proteins) are poorlyimmunogenic. Therefore in the context of therapeutic cancer vaccine, apotent adjuvant is mandatory to be added to the vaccine in order toincrease the level of co-stimulatory molecules on DCs and thereforeaugment the immune system's response to the target antigens. Adjuvantsaccomplish this task by mimicking conserved microbial components thatare naturally recognized by the immune system. They include,lipopolysaccharide (LPS), components of bacterial cell walls, andnucleic acids such as double-stranded RNA (dsRNA), single-stranded DNA(ssDNA), and unmethylated CpG dinucleotide-containing DNA. Theirpresence together with the vaccine can greatly increase the innateimmune response to the antigen. Furthermore, this adjuvant shouldpromote an adaptive immune response with CTLs and type polarized Th1rather than a humoral immune response resulting in antibody production.Different adjuvants have been evaluated, with a limited number havinggained regulatory approval for human use. These include Alum, MPL(monophosphoryl lipid A) and ASO₄ (Alum and MPL) in the US, and MF59(oil-in-water emulsion), ASO₄, liposomes in Europe (Lim, Y. T., Vaccineadjuvant materials for cancer immunotherapy and control of infectiousdisease. Clin Exp Vaccine Res, 2015. 4(1): p. 54-8).

Recently, Toll Like Receptor (TLR) ligands are emerging as promisingclass of adjuvants (Baxevanis, C. N., I. F. Voutsas, and O. E.Tsitsilonis, Toll-like receptor agonists: current status and futureperspective on their utility as adjuvants in improving anticancervaccination strategies. Immunotherapy, 2013. 5(5): p. 497-511). Asignificant development of cancer vaccine studies was thus to includevarious TLR agonists to vaccine formulations, including TLR-3 (polyI:C), TLR-4 (monophosphoryl lipid A; MPL), TLR-5 (flagellin), TLR-7(imiquimod), and TLR-9 (CpG) (Duthie M S, Windish H P, Fox C B, Reed SG. Use of defined TLR ligands as adjuvants within human vaccines.Immunol Rev. 2011; 239:178-196). The types of signaling and cytokinesproduced by immune cells after TLR stimulation control CD4+ T-celldifferentiation into Th1, Th2, Th17, and Treg cells. Stimulation ofimmune cells such as DCs and T cells by most TLR-based adjuvantsproduces proinflammatory cytokines and promotes Th1 and CD8+ T responses(Manicassamy S, Pulendran B. Modulation of adaptive immunity withToll-like receptors. Semin Immunol. 2009; 21:185-193).

Conjugating the vaccine to a TLR ligand is an attractive approach thatoffers several advantages over non-conjugated vaccines including (i)preferential uptake by the immune cells expressing the TLR, (ii) higherimmune response and (iii) reduced risk of inducing peripheral tolerance.Indeed, all the antigen presenting cells loaded with the antigen will besimultaneously activated. Different groups explored this approach withvarious TLR ligands being mainly linked chemically to the peptide orprotein vaccine (Zom G G, Khan S, Filippov D V, Ossendorp F. TLRligand-peptide conjugate vaccines: toward clinical application. AdvImmunol. 2012; 114:177-201). As the chemical linkage to peptide iseasily performed, the most highly investigated TLR ligands for conjugatevaccine are the TLR2 agonist Pam2Cys and Pam3Cys (Fujita, Y. and H.Taguchi, Overview and outlook of Toll-like receptor ligand-antigenconjugate vaccines. Ther Deliv, 2012. 3(6): p. 749-60).

However, to date the majority of cancer vaccines trials have shownlimited efficacy. One explanation is the lack of a therapy that cansimultaneously (i) stimulate multi-epitopic cytotoxic T cell-mediatedimmunity, (ii) induce T_(h) cells and (iii) promote immunologicalmemory. These three parameters are essential to generate potent, longlasting anti-tumor immunity. Indeed, CTLs specific for differentepitopes will allow destruction of more cancer cells within aheterogeneous tumor mass and avoid the outgrowth of antigen-lossvariants (tumor immune escape). T_(h) cells are involved in themaintenance of long-lasting cellular immunity and tumor infiltration byT_(h) cells is also an essential step for the recruitment and functionof CD8⁺ CTLs. Immunological memory is essential to protect against tumorrelapse.

In view of the above, it is the object of the present invention toovercome the drawbacks of current cancer vaccines outlined above and toprovide a novel complex for cancer immunotherapy applicationsrepresenting a more potent vaccine, in particular cancer vaccine, havingimproved anti-tumor activity.

This object is achieved by means of the subject-matter set out below andin the appended claims.

Although the present invention is described in detail below, it is to beunderstood that this invention is not limited to the particularmethodologies, protocols and reagents described herein as these mayvary. It is also to be understood that the terminology used herein isnot intended to limit the scope of the present invention which will belimited only by the appended claims. Unless defined otherwise, alltechnical and scientific terms used herein have the same meanings ascommonly understood by one of ordinary skill in the art.

In the following, the elements of the present invention will bedescribed. These elements are listed with specific embodiments, however,it should be understood that they may be combined in any manner and inany number to create additional embodiments. The variously describedexamples and preferred embodiments should not be construed to limit thepresent invention to only the explicitly described embodiments. Thisdescription should be understood to support and encompass embodimentswhich combine the explicitly described embodiments with any number ofthe disclosed and/or preferred elements. Furthermore, any permutationsand combinations of all described elements in this application should beconsidered disclosed by the description of the present applicationunless the context indicates otherwise.

Throughout this specification and the claims which follow, unless thecontext requires otherwise, the term “comprise”, and variations such as“comprises” and “comprising”, will be understood to imply the inclusionof a stated member, integer or step but not the exclusion of any othernon-stated member, integer or step. The term “consist of” is aparticular embodiment of the term “comprise”, wherein any othernon-stated member, integer or step is excluded. In the context of thepresent invention, the term “comprise” encompasses the term “consistof”. The term “comprising” thus encompasses “including” as well as“consisting” e.g., a composition “comprising” X may consist exclusivelyof X or may include something additional e.g., X+Y.

The terms “a” and “an” and “the” and similar reference used in thecontext of describing the invention (especially in the context of theclaims) are to be construed to cover both the singular and the plural,unless otherwise indicated herein or clearly contradicted by context.Recitation of ranges of values herein is merely intended to serve as ashorthand method of referring individually to each separate valuefalling within the range. Unless otherwise indicated herein, eachindividual value is incorporated into the specification as if it wereindividually recited herein. No language in the specification should beconstrued as indicating any non-claimed element essential to thepractice of the invention.

The word “substantially” does not exclude “completely” e.g., acomposition which is “substantially free” from Y may be completely freefrom Y. Where necessary, the word “substantially” may be omitted fromthe definition of the invention.

The term “about” in relation to a numerical value x means x±10%.

Complexes according to the Present Invention

In a first aspect the present invention provides a complex comprising:

-   -   a) a cell penetrating peptide;    -   b) at least one antigen or antigenic epitope; and    -   c) at least one TLR peptide agonist,

wherein the components a)-c), i.e. the cell penetrating peptide, the atleast one antigen or antigenic epitope and the at least one TLR peptideagonist, are covalently linked.

Such a complex according to the present invention provides simultaneous(i) stimulation of multi-epitopic cytotoxic T cell-mediated immunity,(ii) induction of T_(h) cells and (iii) promotion of immunologicalmemory. Thereby, a complex according to the present invention provides apotent vaccine, in particular having improved anti-tumor activity.

Preferably, the complex according to the present invention is apolypeptide or a protein, in particular a recombinant polypeptide or arecombinant protein, preferably a recombinant fusion protein or arecombinant fusion polypeptide. The term “recombinant” as used hereinmeans that it (here: the polypeptide or the protein) does not occurnaturally. Accordingly, the complex according to the present invention,which is a recombinant polypeptide or a recombinant protein, typicallycomprises components a) to c), wherein components a) to c) are ofdifferent origins, i.e. do not naturally occur in this combination.

In the context of the present invention, i.e. throughout the presentapplication, the terms “peptide”, “polypeptide”, “protein” andvariations of these terms refer to peptide, oligopeptide, oligomer orprotein including fusion protein, respectively, comprising at least twoamino acids joined to each other preferably by a normal peptide bond,or, alternatively, by a modified peptide bond, such as for example inthe cases of isosteric peptides. A peptide, polypeptide or protein canbe composed of L-amino acids and/or D-amino acids. Preferably, apeptide, polypeptide or protein is either (entirely) composed of L-aminoacids or (entirely) of D-amino acids, thereby forming “retro-inversopeptide sequences”. The term “retro-inverso (peptide) sequences” refersto an isomer of a linear peptide sequence in which the direction of thesequence is reversed and the chirality of each amino acid residue isinverted (see e.g. Jameson et al, Nature, 368,744-746 (1994); Brady etal, Nature, 368,692-693 (1994)). In particular, the terms “peptide”,“polypeptide”, “protein also include “peptidomimetics” which are definedas peptide analogs containing non-peptidic structural elements, whichpeptides are capable of mimicking or antagonizing the biologicalaction(s) of a natural parent peptide. A peptidomimetic lacks classicalpeptide characteristics such as enzymatically scissile peptide bonds. Inparticular, a peptide, polypeptide or protein can comprise amino acidsother than the 20 amino acids defined by the genetic code in addition tothese amino acids, or it can be composed of amino acids other than the20 amino acids defined by the genetic code. In particular, a peptide,polypeptide or protein in the context of the present invention canequally be composed of amino acids modified by natural processes, suchas post-translational maturation processes or by chemical processes,which are well known to a person skilled in the art. Such modificationsare fully detailed in the literature. These modifications can appearanywhere in the polypeptide: in the peptide skeleton, in the amino acidchain or even at the carboxy- or amino-terminal ends. In particular, apeptide or polypeptide can be branched following an ubiquitination or becyclic with or without branching. This type of modification can be theresult of natural or synthetic post-translational processes that arewell known to a person skilled in the art. The terms “peptide”,“polypeptide”, “protein” in the context of the present invention inparticular also include modified peptides, polypeptides and proteins.For example, peptide, polypeptide or protein modifications can includeacetylation, acylation, ADP-ribosylation, amidation, covalent fixationof a nucleotide or of a nucleotide derivative, covalent fixation of alipid or of a lipidic derivative, the covalent fixation of aphosphatidylinositol, covalent or non-covalent cross-linking,cyclization, disulfide bond formation, demethylation, glycosylationincluding pegylation, hydroxylation, iodization, methylation,myristoylation, oxidation, proteolytic processes, phosphorylation,prenylation, racemization, seneloylation, sulfatation, amino acidaddition such as arginylation or ubiquitination. Such modifications arefully detailed in the literature (Proteins Structure and MolecularProperties (1993) 2nd Ed., T. E. Creighton, New York; Post-translationalCovalent Modifications of Proteins (1983) B. C. Johnson, Ed., AcademicPress, New York; Seifter et al. (1990) Analysis for proteinmodifications and nonprotein cofactors, Meth. Enzymol. 182: 626-646 andRattan et al., (1992) Protein Synthesis: Post-translationalModifications and Aging, Ann NY Acad Sci, 663: 48-62). Accordingly, theterms “peptide”, “polypeptide”, “protein” preferably include for examplelipopeptides, lipoproteins, glycopeptides, glycoproteins and the like.

However, in a particularly preferred embodiment, the complex accordingto the present invention is a “classical” peptide, polypeptide orprotein, whereby a “classical” peptide, polypeptide or protein istypically composed of amino acids selected from the 20 amino acidsdefined by the genetic code, linked to each other by a normal peptidebond.

If the complex according to the present invention is a polypeptide or aprotein, it is preferred that it comprises at least 50, at least 60, atleast 70, preferably at least 80, at least 90, more preferably at least100, at least 110, even more preferably at least 120, at least 130,particularly preferably at least 140, or most preferably at least 150amino acid residues.

Component a)—Cell Penetrating Peptide

The CPP allows for efficient delivery, i.e. transport and loading, inparticular of at least one antigen or antigenic epitope, into theantigen presenting cells (APCs), in particular into the dendritic cells(DCs) and thus to the dendritic cells' antigen processing machinery.

The term “cell penetrating peptides” (“CPPs”) is generally used todesignate short peptides that are able to transport different types ofcargo molecules across plasma membrane, and, thus, facilitate cellularuptake of various molecular cargoes (from nanosize particles to smallchemical molecules and large fragments of DNA). “Cellularinternalization” of the cargo molecule linked to the cell penetratingpeptide generally means transport of the cargo molecule across theplasma membrane and thus entry of the cargo molecule into the cell.Depending on the particular case, the cargo molecule can, then, bereleased in the cytoplasm, directed to an intracellular organelle, orfurther presented at the cell surface. Cell penetrating ability, orinternalization, of the cell penetrating peptide or complex comprisingsaid cell penetrating peptide, according to the invention can be checkedby standard methods known to one skilled in the art, including flowcytometry or fluorescence microscopy of live and fixed cells,immunocytochemistry of cells transduced with said peptide or complex,and Western blot.

Cell penetrating peptides typically have an amino acid composition thateither contains a high relative abundance of positively charged aminoacids such as lysine or arginine or have a sequence that contains analternating pattern of polar/charged amino acids and non-polar,hydrophobic amino acids. These two types of structures are referred toas polycationic or amphipathic, respectively. Cell-Penetrating peptidesare of different sizes, amino acid sequences, and charges but all CPPshave a common characteristic that is the ability to translocate theplasma membrane and facilitate the delivery of various molecular cargoesto the cytoplasm or to an organelle of a cell. At present, the theoriesof CPP translocation distinguish three main entry mechanisms: directpenetration in the membrane, endocytosis-mediated entry, andtranslocation through the formation of a transitory structure. CPPtransduction is an area of ongoing research. Cell-penetrating peptideshave found numerous applications in medicine as drug delivery agents inthe treatment of different diseases including cancer and virusinhibitors, as well as contrast agents for cell labeling and imaging.

Typically, cell penetrating peptides (CPPs) are peptides of 8 to 50residues that have the ability to cross the cell membrane and enter intomost cell types. Alternatively, they are also called proteintransduction domain (PTDs) reflecting their origin as occurring innatural proteins. Frankel and Pabo simultaneously to Green andLowenstein described the ability of the trans-activating transcriptionalactivator from the human immunodeficiency virus 1 (HIV-TAT) to penetrateinto cells (Frankel, A. D. and C. O. Pabo, Cellular uptake of the tatprotein from human immunodeficiency virus. Cell, 1988. 55(6): p.1189-93). In 1991, transduction into neural cells of the Antennapediahomeodomain (DNA-binding domain) from Drosophila melanogaster wasdescribed (Joliot, A., et al., Antennapedia homeobox peptide regulatesneural morphogenesis. Proc Natl Acad Sci USA, 1991. 88(5): p. 1864-8).In 1994, the first 16-mer peptide CPP called Penetratin, having theamino acid sequence RQIKIYFQNRRMKWKK (SEQ ID NO: 1) was characterizedfrom the third helix of the homeodomain of Antennapedia (Derossi, D., etal., The third helix of the Antennapedia homeodomain translocatesthrough biological membranes. J Biol Chem, 1994. 269(14): p. 10444-50),followed in.1998 by the identification of the minimal domain of TAT,having the amino acid sequence YGRKKRRQRRR (SEQ ID NO: 2) required forprotein transduction (Vives, E., P. Brodin, and B. Lebleu, A truncatedHIV-1 Tat protein basic domain rapidly translocates through the plasmamembrane and accumulates in the cell nucleus. J Biol Chem, 1997.272(25): p. 16010-7). Over the past two decades, dozens of peptides weredescribed from different origins including viral proteins, e.g. VP22(Elliott, G. and P. O′Hare, Intercellular trafficking and proteindelivery by a herpesvirus structural protein. Cell, 1997. 88(2): p.223-33) and ZEBRA (Rothe, R., et al., Characterization of thecell-penetrating properties of the Epstein-Barr virus ZEBRAtrans-activator. J Biol Chem, 2010. 285(26): p. 20224-33), or fromvenoms, e.g. melittin (Dempsey, C. E., The actions of melittin onmembranes. Biochim Biophys Acta, 1990. 1031(2): p. 143-61), mastoporan(Konno, K., et al., Structure and biological activities of eumeninemastoparan-AF (EMP-AF), a new mast cell degranulating peptide in thevenom of the solitary wasp (Anterhynchium flavomarginatum micado).Toxicon, 2000. 38(11): p. 1505-15), maurocalcin (Esteve, E., et al.,Transduction of the scorpion toxin maurocalcine into cells. Evidencethat the toxin crosses the plasma membrane. J Biol Chem, 2005. 280(13):p. 12833-9), crotamine (Nascimento, F. D., et al., Crotamine mediatesgene delivery into cells through the binding to heparan sulfateproteoglycans. J Biol Chem, 2007. 282(29): p. 21349-60) or buforin(Kobayashi, S., et al., Membrane translocation mechanism of theantimicrobial peptide buforin 2. Biochemistry, 2004. 43(49): p.15610-6). Synthetic CPPs were also designed including the poly-arginine(R8, R9, R10 and R12) (Futaki, S., et al., Arginine-rich peptides. Anabundant source of membrane-permeable peptides having potential ascarriers for intracellular protein delivery. J Biol Chem, 2001. 276(8):p. 5836-40) or transportan (Pooga, M., et al., Cell penetration bytransportan. FASEB J, 1998. 12(1): p. 67-77). Any of the above describedCPPs may be used as cell penetrating peptide, i.e. as component a), inthe complex according to the present invention. In particular, thecomponent a), i.e. the CPP, in the complex according to the presentinvention may comprise the minimal domain of TAT, having the amino acidsequence YGRKKRRQRRR (SEQ ID NO: 2). In particular, the component a),i.e. the CPP, in the complex according to the present invention maycomprise Penetratin having the amino acid sequence RQIKIYFQNRRMKWKK (SEQID NO: 1).

Various CPPs, which can be used as cell penetrating peptide, i.e. ascomponent a), in the complex according to the present invention, arealso disclosed in the review: Milletti, F., Cell-penetrating peptides:classes, origin, and current landscape. Drug Discov Today 17 (15-16):850-60, 2012. In other words, the CPPs disclosed in Milletti, F., 2012,Cell-penetrating peptides: classes, origin, and current landscape. DrugDiscov Today 17 (15-16): 850-60 can be used as cell penetrating peptide,i.e. as component a), in the complex according to the present invention.This includes in particular cationic CPPs, amphipatic CPPs, andhydrophobic CPPs as well as CPPs derived from heparan-, RNA- andDNA-binding proteins (cf. Table 1 of Milletti, F., Cell-penetratingpeptides: classes, origin, and current landscape. Drug Discov Today 17(15-16): 850-60, 2012), CPPs derived from signal peptides (cf. Table 2of Milletti, F., Cell-penetrating peptides: classes, origin, and currentlandscape. Drug Discov Today 17 (15-16): 850-60, 2012), CPPs derivedfrom antimicrobial peptides (cf. Table 3 of Milletti, F.,Cell-penetrating peptides: classes, origin, and current landscape. DrugDiscov Today 17 (15-16): 850-60, 2012), CPPs derived from viral proteins(cf. Table 4 of Milletti, F., Cell-penetrating peptides: classes,origin, and current landscape. Drug Discov Today 17 (15-16): 850-60,2012), CPPs derived from various natural proteins (cf. Table 5 ofMilletti, F., Cell-penetrating peptides: classes, origin, and currentlandscape. Drug Discov Today 17 (15-16): 850-60, 2012), and DesignedCPPs and CPPs derived from peptide libraries (cf. Table 6 of Milletti,F., Cell-penetrating peptides: classes, origin, and current landscape.Drug Discov Today 17 (15-16): 850-60, 2012).

Preferably, the cell penetrating peptide, which is comprised by thecomplex according to the present invention,

-   i) has a length of the amino acid sequence of said peptide of 5 to    50 amino acids in total, preferably of 10 to 45 amino acids in    total, more preferably of 15 to 45 amino acids in total; and/or-   ii) has an amino acid sequence comprising a fragment of the minimal    domain of ZEBRA, said minimal domain extending from residue 170 to    residue 220 of the ZEBRA amino acid sequence according to SEQ ID NO:    3, wherein, optionally, 1, 2, 3, 4, or 5 amino acids have been    substituted, deleted, and/or added without abrogating said peptide's    cell penetrating ability, or a sequence variant of such a fragment.

Thereby, it is preferred that the cell penetrating peptide, which iscomprised by the complex according to the present invention,

-   i) has a length of the amino acid sequence of said peptide of 5 to    50 amino acids in total, preferably of 10 to 45 amino acids in    total, more preferably of 15 to 45 amino acids in total; and-   ii) has an amino acid sequence comprising a fragment of the minimal    domain of ZEBRA, said minimal domain extending from residue 170 to    residue 220 of the ZEBRA amino acid sequence according to SEQ ID NO:    3, wherein, optionally, 1, 2, 3, 4, or 5 amino acids have been    substituted, deleted, and/or added without abrogating said peptide's    cell penetrating ability, or a sequence variant of such a fragment.

Such preferred CPPs are disclosed in WO 2014/041505.

The term “ZEBRA” (also known as Zta, Z, EB1, or BZLF1) generally meansthe basic-leucine zipper (bZIP) transcriptional activator of theEpstein-Barr virus (EBV). The minimal domain of ZEBRA, which exhibitscell penetrating properties, has been identified as spanning fromresidue 170 to residue 220 of ZEBRA. The amino acid sequence of ZEBRA isdisclosed under NCBI accession number YP_401673 and comprises 245 aminoacids represented in SEQ ID NO: 3:

MMDPNSTSEDVKFTPDPYQVPFVQAFDQATRVYQDLGGPSQAPLPCVLWPVLPEPLPQGQLTAYHVSTAPTGSWFSAPQPAPENAYQAYAAPQLFPVSDITQNQQTNQAGGEAPQPGDNSTVQTAAAVVFACPGANQGQQLADIGVPQPAPVAAPARRTRKPQQPESLEECDSELEIKRYKNRVASRKCRAKFKQLLQHYREVAAAKSSENDRLRLLLKQMCPSLDVDSIIPRTPDVLHEDLLNF (SEQ IDNO: 3—ZEBRA amino acid sequence (natural sequence from Epstein-Barrvirus (EBV)) (YP_401673))

Recently, a CPP derived from the viral protein ZEBRA was described totransduce protein cargoes across biological membranes by both (i) directtranslocation and (ii) lipid raft-mediated endocytosis (Rothe R, LiguoriL, Villegas-Mendez A, Marques B, Grunwald D, Drouet E, et al.Characterization of the cell-penetrating properties of the Epstein-Barrvirus ZEBRA trans-activator. The Journal of biological chemistry 2010;285(26):20224-33). The present inventors assume that these twomechanisms of entry should promote both MHC class I and II restrictedpresentation of cargo antigens to CD8⁺ and CD4⁺ T cells, respectively.Accordingly, such a CPP can deliver multi-epitopic peptides to dendriticcells (DCs), and subsequently to promote CTL and Th cell activation andanti-tumor function. Such a CPP can thus efficiently deliver the complexaccording to the present invention to antigen presenting cells (APCs)and lead to multi-epitopic MHC class I and II restricted presentation.

In the context of the present invention, the term “MHC class I”designates one of the two primary classes of the MajorHistocompatibility Complex molecules. The MHC class I (also noted “MHCI”) molecules are found on every nucleated cell of the body. Thefunction of MHC class I is to display an epitope to cytotoxic cells(CTLs). In humans, MHC class I molecules consist of two polypeptidechains, α- and β2-microglobulin (b2m). Only the α chain is polymorphicand encoded by a HLA gene, while the b2m subunit is not polymorphic andencoded by the Beta-2 microglobulin gene. In the context of the presentinvention, the term “MHC class II” designates the other primary class ofthe Major Histocompatibility Complex molecules. The MHC class II (alsonoted “MHC II”) molecules are found only on a few specialized celltypes, including macrophages, dendritic cells and B cells, all of whichare dedicated antigen-presenting cells (APCs).

Preferably, the sequence variant of a fragment of the minimal domain ofZEBRA as described above shares, in particular over the whole length, atleast 70%, at least 75%, preferably at least 80%, more preferably atleast 85%, even more preferably at least 90%, particularly preferably atleast 95%, most preferably at least 99% amino acid sequence identitywith the fragment of the minimal domain of ZEBRA as described abovewithout abrogating the cell penetrating ability of the cell penetratingpeptide. In particular, a “fragment” of the minimal domain of ZEBRA asdefined above is preferably to be understood as a truncated sequencethereof, i.e. an amino acid sequence, which is N-terminally,C-terminally and/or intrasequentially truncated compared to the aminoacid sequence of the native sequence. Moreover, such a “fragment” of theminimal domain of ZEBRA has preferably a length of 5 to 50 amino acidsin total, preferably of 10 to 45 amino acids in total, more preferablyof 15 to 45 amino acids in total.

Accordingly, the term “sequence variant” as used in the context of thepresent invention, i.e. throughout the present application, refers toany alteration in a reference sequence. The term “sequence variant”includes nucleotide sequence variants and amino acid sequence variants.Preferably, a reference sequence is any of the sequences listed in the“Table of Sequences and SEQ ID Numbers” (Sequence listing), i.e. SEQ IDNO: 1 to SEQ ID NO: 79. Preferably, a sequence variant shares, inparticular over the whole length of the sequence, at least 70%, at least75%, preferably at least 80%, more preferably at least 85%, even morepreferably at least 90%, particularly preferably at least 95%, mostpreferably at least 99% sequence identity with a reference sequence,whereby sequence identity is calculated as described below. Inparticular, a sequence variant preserves the specific function of thereference sequence. Sequence identity is calculated as described below.In particular, an amino acid sequence variant has an altered sequence inwhich one or more of the amino acids in the reference sequence isdeleted or substituted, or one or more amino acids are inserted into thesequence of the reference amino acid sequence. As a result of thealterations, the amino acid sequence variant has an amino acid sequencewhich is at least 70%, at least 75%, preferably at least 80%, morepreferably at least 85%, even more preferably at least 90%, particularlypreferably at least 95%, most preferably at least 99% identical to thereference sequence. For example, variant sequences which are at least90% identical have no more than 10 alterations, i.e. any combination ofdeletions, insertions or substitutions, per 100 amino acids of thereference sequence.

In the context of the present invention, an amino acid sequence “sharinga sequence identity” of at least, for example, 95% to a query amino acidsequence of the present invention, is intended to mean that the sequenceof the subject amino acid sequence is identical to the query sequenceexcept that the subject amino acid sequence may include up to five aminoacid alterations per each 100 amino acids of the query amino acidsequence. In other words, to obtain an amino acid sequence having asequence of at least 95% identity to a query amino acid sequence, up to5% (5 of 100) of the amino acid residues in the subject sequence may beinserted or substituted with another amino acid or deleted, preferablywithin the above definitions of variants or fragments. The same, ofcourse, also applies similarly to nucleic acid sequences.

For (amino acid or nucleic acid) sequences without exact correspondence,a “% identity” of a first sequence may be determined with respect to asecond sequence. In general, these two sequences to be compared arealigned to give a maximum correlation between the sequences. This mayinclude inserting “gaps” in either one or both sequences, to enhance thedegree of alignment. A % identity may then be determined over the wholelength of each of the sequences being compared (so-called globalalignment), that is particularly suitable for sequences of the same orsimilar length, or over shorter, defined lengths (so-called localalignment), that is more suitable for sequences of unequal length.

Methods for comparing the identity and homology of two or more sequencesare well known in the art. The percentage to which two sequences areidentical can e.g. be determined using a mathematical algorithm. Apreferred, but not limiting, example of a mathematical algorithm whichcan be used is the algorithm of Karlin et al. (1993), PNAS USA,90:5873-5877. Such an algorithm is integrated in the BLAST family ofprograms, e.g. BLAST or NBLAST program (see also Altschul et al., 1990,J. Mol. Biol. 215, 403-410 or Altschul et al. (1997), Nucleic Acids Res,25:3389-3402), accessible through the home page of the NCBI at worldwide web site ncbi.nlm.nih.gov) and FASTA (Pearson (1990), MethodsEnzymol. 183, 63-98; Pearson and Lipman (1988), Proc. Natl. Acad. Sci.U.S.A 85, 2444-2448.). Sequences which are identical to other sequencesto a certain extent can be identified by these programmes. Furthermore,programs available in the Wisconsin Sequence Analysis Package, version9.1 (Devereux et al., 1984, Nucleic Acids Res., 387-395), for examplethe programs BESTFIT and GAP, may be used to determine the % identitybetween two polynucleotides and the identity and the % homology oridentity between two polypeptide sequences. BESTFIT uses the “localhomology” algorithm of (Smith and Waterman (1981), J. Mol. Biol. 147,195-197.) and finds the best single region of similarity between twosequences.

More preferably, the fragments of the cell penetrating peptide accordingto the invention or the variants thereof as described above furtherretain said peptide's ability to present a cargo molecule such asantigens or antigenic epitopes at the surface of a cell, such as anantigen-presenting cell, in the context of MHC class I and/or MHC classII molecules. The ability of a cell penetrating peptide or complexcomprising said cell penetrating peptide to present a cargo moleculesuch as antigens or antigenic epitopes at the surface of a cell in thecontext of MHC class I and/or MHC class II molecules can be checked bystandard methods known to one skilled in the art, including capacity tostimulate proliferation and/or function of MHC-restricted CD4⁺ or CD8⁺ Tcells with specificity for these epitopes.

The preferred cell penetrating peptide, which

-   i) has a length of the amino acid sequence of said peptide of 5 to    50 amino acids in total, preferably of 10 to 45 amino acids in    total, more preferably of 15 to 45 amino acids in total; and/or-   ii) has an amino acid sequence comprising a fragment of the minimal    domain of ZEBRA, said minimal domain extending from residue 170 to    residue 220 of the ZEBRA amino acid sequence according to SEQ ID NO:    3, wherein, optionally, 1, 2, 3, 4, or 5 amino acids have been    substituted, deleted, and/or added without abrogating said peptide's    cell penetrating ability, or a variant of such a fragment

preferably comprises an amino acid sequence having at least oneconservatively substituted amino acid compared to the referencedsequence, meaning that a given amino acid residue is replaced by aresidue having similar physiochemical characteristics.

Generally, substitutions for one or more amino acids present in thereferenced amino acid sequence should be made conservatively. Examplesof conservative substitutions include substitution of one aliphaticresidue for another, such as Ile, Val, Leu, or Ala for one another, orsubstitutions of one polar residue for another, such as between Lys andArg; Glu and Asp; or Gln and Asn. Other such conservative substitutions,for example, substitutions of entire regions having similarhydrophobicity properties, are well known (Kyte and Doolittle, 1982, J.Mol. Biol. 157(1):105-132). Substitutions of one or more L-amino acidswith one or more D-amino acids are to be considered as conservativesubstitutions in the context of the present invention. Exemplary aminoacid substitutions are presented in Table 1 below:

TABLE 1 Original residues Examples of substitutions Ala (A) Val, Leu,Ile, Gly Arg (R) His, Lys Asn (N) Gln Asp (D) Glu Cys (C) Ser Gln (Q)Asn Glu (E) Asp Gly (G) Pro, Ala His (H) Lys, Arg Ile (I) Leu, Val, Met,Ala, Phe Leu (L) Ile, Val, Met, Ala, Phe Lys (K) Arg, His Met (M) Leu,Ile, Phe Phe (F) Leu, Val, Ile, Tyr, Trp, Met Pro (P) Ala, Gly Ser (S)Thr Thr (T) Ser Trp (W) Tyr, Phe Tyr (Y) Trp, Phe Val (V) Ile, Met, Leu,Phe, Ala

Particularly preferably, the preferred cell penetrating peptide, which

-   i) has a length of the amino acid sequence of said peptide of 5 to    50 amino acids in total, preferably of 10 to 45 amino acids in    total, more preferably of 15 to 45 amino acids in total; and/or-   ii) has an amino acid sequence comprising a fragment of the minimal    domain of ZEBRA, said minimal domain extending from residue 170 to    residue 220 of the ZEBRA amino acid sequence according to SEQ ID NO:    3, wherein, optionally, 1, 2, 3, 4, or 5 amino acids have been    substituted, deleted, and/or added without abrogating said peptide's    cell penetrating ability, or a variant of such a fragment

comprises a Cys (C) substituted into a Ser (S), at the equivalent ofposition 189 relative to ZEBRA amino acid sequence of SEQ ID NO: 3.

Thereby, it is preferred that such a preferred cell penetrating peptidehas an amino acid sequence comprising a sequence according to thefollowing general formula (I):

X₁X₂X₃X₄X₅X₆X₇X₈X₉X₁₀X₁₁SX₁₃X₁₄X₁₅X₁₆X₁₇

with 0, 1, 2, 3, 4, or 5 amino acids which are substituted, deleted,and/or added without abrogating said peptide's cell penetrating ability,wherein

-   -   X₁ is K, R, or H, preferably X₁ is K or R;    -   X₂ is R, K, or H, preferably X₂ is R or K;    -   X₃ is Y, W, or F, preferably X₃ is Y, W, or F;    -   X₄ is K, R, or H, preferably X₄ is K or R;    -   X₅ is N or Q;    -   X₆ is R, K, or H, preferably X₆ is R or K;    -   X₇ is V, I, M, L, F, or A, preferably X7 is V, I, M or L;    -   X₈ is A, V, L, I, or G, preferably X₈ is A or G;    -   X₉ is S or T;    -   X₁₀ is R, K, or H, preferably X₁₀ is R or K;    -   X₁₁ is K, R, or H, preferably X₁₁ is K or R;    -   X₁₃ is R, K, or H, preferably X₁₃ is R or K;    -   X₁₄ is A, V, L, I, or G, preferably X₁₄ is A or G;    -   X₁₅ is K, R, or H, preferably X₁₅ is K or R;    -   X₁₆ is F, L, V, I, Y, W, or M, preferably X₁₆ is F, Y or W; and    -   X₁₇ is K, R, or H, preferably X₁₇ is K or R.

Preferably, such a peptide, polypeptide or protein is either (entirely)composed of L-amino acids or (entirely) of D-amino acids, therebyforming “retro-inverso peptide sequences”. The term “retro-inverso(peptide) sequences” refers to an isomer of a linear peptide sequence inwhich the direction of the sequence is reversed and the chirality ofeach amino acid residue is inverted (see e.g. Jameson et al., Nature,368,744-746 (1994); Brady et al., Nature, 368,692-693 (1994)).

In a particular embodiment, the cell penetrating peptide according tothe invention is as generically defined above by general formula (I),wherein X₁ is K.

In a particular embodiment, the cell penetrating peptide according tothe invention is as generically defined above by general formula (I),wherein X₂ is R.

In a particular embodiment, the cell penetrating peptide according tothe invention is as generically defined above by general formula (I),wherein X₃ is Y.

In a particular embodiment, the cell penetrating peptide according tothe invention is as generically defined above by general formula (I),wherein X₄ is K.

In a particular embodiment, the cell penetrating peptide according tothe invention is as generically defined above by general formula (I),wherein X₅ is N.

In a particular embodiment, the cell penetrating peptide according tothe invention is as generically defined above by general formula (I),wherein X₆ is R.

In a particular embodiment, the cell penetrating peptide according tothe invention is as generically defined above by general formula (I),wherein X₇ is V.

In a particular embodiment, the cell penetrating peptide according tothe invention is as generically defined above by general formula (I),wherein X₈ is A.

In a particular embodiment, the cell penetrating peptide according tothe invention is as generically defined above by general formula (I),wherein X₉ is S.

In a particular embodiment, the cell penetrating peptide according tothe invention is as generically defined above by general formula (I),wherein X₁₀ is R.

In a particular embodiment, the cell penetrating peptide according tothe invention is as generically defined above by general formula (I),wherein X₁₁ is K.

In a particular embodiment, the cell penetrating peptide according tothe invention is as generically defined above by general formula (I),wherein X₁₃ is R.

In a particular embodiment, the cell penetrating peptide according tothe invention is as generically defined above by general formula (I),wherein X₁₄ is A.

In a particular embodiment, the cell penetrating peptide according tothe invention is as generically defined above by general formula (I),wherein X₁₅ is K.

In a particular embodiment, the cell penetrating peptide according tothe invention is as generically defined above by general formula (I),wherein X₁₆ is F.

In a particular embodiment, the cell penetrating peptide according tothe invention is as generically defined above by general formula (I),wherein X₁₇ is K.

In a particular embodiment, the cell penetrating peptide according tothe invention is as generically defined above by general formula (I),wherein the amino acid at position equivalent to position 12 relative togeneral formula (I) is a Ser (S).

It is also particularly preferred, that the preferred cell penetratingpeptide, which

-   i) has a length of the amino acid sequence of said peptide of 5 to    50 amino acids in total, preferably of 10 to 45 amino acids in    total, more preferably of 15 to 45 amino acids in total; and/or-   ii) has an amino acid sequence comprising a fragment of the minimal    domain of ZEBRA, said minimal domain extending from residue 170 to    residue 220 of the ZEBRA amino acid sequence according to SEQ ID NO:    3, wherein, optionally, 1, 2, 3, 4, or 5 amino acids have been    substituted, deleted, and/or added without abrogating said peptide's    cell penetrating ability, or a variant of such a fragment

comprises or consists of an amino acid sequence selected from the groupconsisting of amino acid sequences according to SEQ ID NO: 4-13, orsequence variants thereof without abrogating said peptide's cellpenetrating ability, preferably sequence variants having 0, 1, 2, 3, 4,or 5 amino acids substituted, deleted and/or added without abrogatingsaid peptide's cell penetrating ability.

CPP1 (Z11): (SEQ ID NO: 4) KRYKNRVASRKCRAKFKQLLQHYREVAAAKSSENDRLRLLLKQMCCPP2 (Z12): (SEQ ID NO: 5) KRYKNRVASRKCRAKFKQLLQHYREVAAAKSSENDRLRLLLKCPP3 (Z13): (SEQ ID NO: 6) KRYKNRVASRKSRAKFKQLLQHYREVAAAKSSENDRLRLLLKCPP4 (Z14): (SEQ ID NO: 7) KRYKNRVASRKSRAKFKQLLQHYREVAAAK CPP5 (Z15):(SEQ ID NO: 8) KRYKNRVASRKSRAKFK CPP6 (Z16): (SEQ ID NO: 9)QHYREVAAAKSSEND CPP7 (Z17): (SEQ ID NO: 10) QLLQHYREVAAAK CPP8 (Z18):(SEQ ID NO: 11) REVAAAKSS END RLRLLLK CPP9 (Z19): (SEQ ID NO: 12)KRYKNRVA CPP10 (Z20): (SEQ ID NO: 13) VASRKSRAKFK

Thereby, a cell penetrating peptide is particularly preferred, which hasan amino acid sequence comprising or consisting of an amino acidsequence according to SEQ ID NO: 6 (CPP3/Z13), SEQ ID NO: 7 (CPP4/Z14),SEQ ID NO: 8 (CPP5/Z15), or SEQ ID NO: 11 (CPPB/Z18), or sequencevariants thereof without abrogating said peptide's cell penetratingability, preferably sequence variants having 0, 1, 2, 3, 4, or 5 aminoacids substituted, deleted and/or added without abrogating saidpeptide's cell penetrating ability. Moreover, a cell penetrating peptideis more preferred, which has an amino acid sequence comprising orconsisting of an amino acid sequence according to SEQ ID NO: 6(CPP3/Z13) or SEQ ID NO: 7 (CPP4/Z14) or sequence variants thereofwithout abrogating said peptide's cell penetrating ability, preferablysequence variants having 0, 1, 2, 3, 4, or 5 amino acids substituted,deleted and/or added without abrogating said peptide's cell penetratingability. Moreover, a cell penetrating peptide is most preferred, whichhas an amino acid sequence comprising or consisting of an amino acidsequence according to SEQ ID NO: 6 (CPP3/Z13) or sequence variantsthereof without abrogating said peptide's cell penetrating ability,preferably sequence variants having 0, 1, 2, 3, 4, or 5 amino acidssubstituted, deleted and/or added without abrogating said peptide's cellpenetrating ability.

In one preferred embodiment, the cell penetrating peptide according tothe invention has an amino acid sequence comprising or consisting of SEQID NO: 6 (CPP3/Z13).

In another preferred embodiment, the cell penetrating peptide accordingto the invention has an amino acid sequence comprising or consisting ofSEQ ID NO: 7 (CPP4/Z14).

In another preferred embodiment, the cell penetrating peptide accordingto the invention has an amino acid sequence comprising or consisting ofSEQ ID NO: 8 (CPP5/Z15).

In another preferred embodiment, the cell penetrating peptide accordingto the invention has an amino acid sequence comprising or consisting ofSEQ ID NO: 11 (CPPB/Z18).

It will be understood by one skilled in the art that the primary aminoacid sequence of the cell penetrating peptide of the invention mayfurther be post-translationally modified, such as by glycosylation orphosphorylation, without departing from the invention.

In a further embodiment, the cell penetrating peptide according to theinvention optionally further comprises, in addition to its amino acidsequence as described above, any one of, or any combination of:

-   -   (i) a nuclear localization signal (NLS). Such signals are well        known to the skilled person and are described in Nair et al.        (2003, Nucleic Acids Res. 37(7): 397-399)    -   (ii) a targeting peptide, including tumor homing peptides such        as those described in Kapoor et al. (2072, PLoS ONE 7(4):        e35187) and listed in        http://crdd.osdd.net/raghava/tumorhope/general.php?

Preferably, the cell penetrating peptide according to the invention islinked to an antigen or antigenic epitope and facilitates the cellularinternalization of said antigen or antigenic epitope.

The complex according to the present invention may comprise one singlecell penetrating peptide or more than one cell penetrating peptides.Preferably, the complex according to the present invention comprises nomore than five cell penetrating peptides, more preferably the complexaccording to the present invention comprises no more than four cellpenetrating peptides, even more preferably the complex according to thepresent invention comprises no more than three cell penetratingpeptides, particularly preferably the complex according to the presentinvention comprises no more than two cell penetrating peptides and mostpreferably the complex according to the present invention comprises onesingle cell penetrating peptide.

Component b)—Antigen/Antigenic Epitope

The complex according to the present invention comprises as component b)at least one antigen or antigenic epitope.

As used herein, an “antigen” is any structural substance which serves asa target for the receptors of an adaptive immune response, in particularas a target for antibodies, T cell receptors, and/or B cell receptors.An “epitope”, also known as “antigenic determinant”, is the part (orfragment) of an antigen that is recognized by the immune system, inparticular by antibodies, T cell receptors, and/or B cell receptors.Thus, one antigen has at least one epitope, i.e. a single antigen hasone or more epitopes. In the context of the present invention, the term“epitope” is mainly used to designate T cell epitopes, which arepresented on the surface of an antigen-presenting cell, where they arebound to Major Histocompatibility Complex (MHC). T cell epitopespresented by MHC class I molecules are typically, but not exclusively,peptides between 8 and 11 amino acids in length, whereas MHC class IImolecules present longer peptides, generally, but not exclusively,between 12 and 25 amino acids in length.

Preferably, in the complex according to the present invention, the atleast one antigen or antigenic epitope is selected from the groupconsisting of: (i) a peptide, a polypeptide, or a protein, (ii) apolysaccharide, (iii) a lipid, (iv) a lipoprotein or a lipopeptide, (v)a glycolipid, (vi) a nucleic acid, and (vii) a small molecule drug or atoxin. Thus, the at least one antigen or antigenic epitope may be apeptide, a protein, a polysaccharide, a lipid, a combination thereofincluding lipoproteins and glycolipids, a nucleic acid (e.g. DNA, siRNA,shRNA, antisense oligonucleotides, decoy DNA, plasmid), or a smallmolecule drug (e.g. cyclosporine A, paclitaxel, doxorubicin,methotrexate, 5-aminolevulinic acid), or any combination thereof inparticular if more than one antigen or antigenic epitope is comprised bythe inventive complex.

It is understood that the at least one antigen or antigenic epitope cancomprise for example at least one, i.e. one or more, peptides,polypeptides or proteins linked together and/or at least one, i.e. oneor more, nucleic acids, e.g. where each one encodes one peptide orpolypeptide. Also the at least one antigen or antigenic epitope can be acombination of a protein, a lipid, and/or a polysaccharide includinglipoproteins and glycolipids. Thus, in particular if the complexaccording to the present invention comprises more than one antigen orantigenic epitope, it can comprise more than one peptide, polypeptide,or protein, more than one polysaccharide, more than one lipid, more thanone lipoprotein, more than one glycolipid, more than one nucleic acid,more than one small molecule drug or toxin, or a combination thereof.

Preferably, the complex according to the invention comprises at leastone antigen or antigenic epitope comprising one or more epitope(s) froma cancer/tumor-associated antigen, a cancer/tumor-specific antigen,and/or an antigenic protein from a pathogen, including viral, bacterial,fungal, protozoal and multicellular parasitic antigenic protein.

More preferably, the at least one antigen or antigenic epitope comprisesor consists of (i) at least one pathogen epitope and/or (ii) at leastone cancer/tumor epitope, in particular at least one tumor epitope. Mostpreferably, the at least one antigen or antigenic epitope comprises orconsists of at least one cancer/tumor epitope, in particular at leastone tumor epitope.

It is particularly preferred that the complex according to the presentinvention comprises only such antigen(s) or antigenic epitope(s), whichare cancer/tumor-associated antigen(s), cancer/tumor-specific antigen(s)and/or cancer/tumor epitope(s); in particular, which aretumor-associated antigen(s), tumor-specific antigen(s), and/or tumorepitope(s).

As used herein, “cancer epitope” means an epitope from acancer-associated antigen or from a cancer-specific antigen.Accordingly, “tumor epitope” means an epitope from a tumor-associatedantigen or from a tumor-specific antigen. Such epitopes are typicallyspecific (or associated) for a certain kind of cancer/tumor. Forinstance, cancer/tumor epitopes include glioma epitopes. In particular,cancer/tumor-associated (also cancer/tumor-related) antigens areantigens, which are expressed by both, cancer/tumor cells and normalcells. Accordingly, those antigens are normally present since birth (oreven before). Accordingly, there is a chance that the immune systemdeveloped self-tolerance to those antigens. Cancer/tumor-specificantigens, in contrast, are antigens, which are expressed specifically bycancer/tumor cells, but not by normal cells. Cancer/tumor-specificantigens include in particular neoantigens. In general neoantigens areantigens, which were not present before and are, thus, “new” to theimmune system. Neoantigens are typically due to somatic mutations. Inthe context of cancer/tumors, cancer/tumor-specific neoantigens weretypically not present before the cancer/tumor developed andcancer/tumor-specific neoantigens are usually encoded by somatic genemutations in the cancerous cells/tumor cells. Since neoantigens are newto the immune system, the risk of self-tolerance of those antigens isconsiderably lower as compared to cancer/tumor-associated antigens.However, every cancer's set of tumor-specific mutations appears to beunique. Accordingly, in the context of the present invention it ispreferred that such cancer/tumor-specific antigens, in particularneoantigens, are identified in a subject diagnosed with a cancer bymethods known to the skilled person, e.g., cancer genome sequencing.After identification, the respective cancer/tumor-specific neoantigensand/or cancer/tumor-specific neoantigenic epitopes are used in a complexaccording to the present invention.

Preferably, a complex according to the present invention comprises oneor more cancer/tumor-associated epitopes and/or one or morecancer/tumor-associated antigens (but preferably nocancer/tumor-specific epitopes). It is also preferred that a complexaccording to the present invention comprises one or morecancer/tumor-specific epitopes and/or one or more cancer/tumor-specificantigens (but preferably no cancer/tumor-associated epitopes). A complexaccording to the present invention may also preferably comprise both,(i) one or more cancer/tumor-associated epitopes and/or one or morecancer/tumor-associated antigens and (ii) one or morecancer/tumor-specific epitopes and/or one or more cancer/tumor-specificantigens.

Suitable cancer/tumor epitopes can be retrieved for example fromcancer/tumor epitope databases, e.g. from van der Bruggen P, StroobantV, Vigneron N, Van den Eynde B. Peptide database: T cell-defined tumorantigens. Cancer Immun 2013; URL:http://www.cancerimmunity.org/peptide/, wherein human tumor antigensrecognized by CD4+ or CD8+ T cells are classified into four major groupson the basis of their expression pattern, or from the database“Tantigen” (TANTIGEN version 1.0, Dec. 1, 2009; developed byBioinformatics Core at Cancer Vaccine Center, Dana-Farber CancerInstitute; URL: http://cvc.dfci.harvard.edu/tadb/). Examples ofcancer/tumor epitopes include e.g. TRP2-derived epitopes, glycoprotein100 (gp100) melanoma antigen-derived epitopes, glycoprotein 70 (gp70)antigen-derived epitopes, survivin epitopes, IEa epitopes, IL13rα2,Epha2 (ephrin type-A receptor 2), immunogenic fragments thereof, andfusions of such antigens and/or fragments. Furthermore, examples ofcancer/tumor epitopes include epitopes of neoantigens, such as, forexample, a neoantigen from MC-38 tumor cell line as described by Yadavet al. Nature. 2014 Nov. 27; 515(7528):572-6. As described above,neoantigens are antigens, which are entirely absent from the normalhuman genome. As compared with nonmutated self-antigens, neoantigens areof relevance to tumor control, as the quality of the T cell pool that isavailable for these antigens is not affected by central T celltolerance. In particular, neoantigens may be based on individual tumorgenomes. Potential neoantigens may be predicted by methods known to theskilled person, such as cancer genome sequencing or deep-sequencingtechnologies identifying mutations within the protein-coding part of the(cancer) genome.

Specific examples of cancer/tumor-associated, in particulartumor-related, or tissue-specific antigens useful in a complex accordingto the present invention include, but are not limited to, the followingantigens: Prostate: prostate-specific antigen (PSA), prostate-specificmembrane antigen (PSMA), PAP, PSCA (PNAS 95(4) 1735-1740 1998), prostatemucin antigen (PMA) (Beckett and Wright, 1995, Int. J. Cancer 62:703-710), Prostase, Her-2neu, SPAS-1; Melanoma: TRP-2, tyrosinase, MelanA/Mart-1, gplOO, BAGE, GAGE, GM2 ganglioside; Breast: Her2-neu, kinesin2, TATA element modulatory factor 1, tumor protein D52, MAGE D, ING2,HIP-55, TGF-1 anti-apoptotic factor, HOM-Mel-40/SSX2, epithelial antigen(LEA 135), DF31MUC1 antigen (Apostolopoulos et al., 1996 Immunol. Cell.Biol. 74: 457-464; Pandey et al., 1995, Cancer Res. 55: 4000-4003);Testis: MAGE-1, HOM-Me1-40/SSX2, NY-ESO-1; Colorectal: EGFR, CEA; Lung:MAGE D, EGFR Ovarian Her-2neu; Baldder: transitional cell carcinoma(TCC) (Jones et al., 1997, Anticancer Res. 17: 685-687), Severalcancers: Epha2, Epha4, PCDGF, HAAH, Mesothelin; EPCAM; NY-ESO-1,glycoprotein MUC1 and NIUC10 mucins p5 (especially mutated versions),EGER; Miscellaneous tumor: cancer-associated serum antigen (CASA) andcancer antigen 125 (CA 125) (Kierkegaard et al., 1995, Gynecol. Oncol.59: 251-254), the epithelial glycoprotein 40 (EGP40) (Kievit et al.,1997, Int. J. Cancer 71: 237-245), squamous cell carcinoma antigen (SCC)(Lozza et al., 1997 Anticancer Res. 17: 525-529), cathepsin E (Mota etal., 1997, Am. J Pathol. 150: 1223-1229), tyrosinase in melanoma(Fishman et al., 1997 Cancer 79: 1461-1464), cell nuclear antigen (PCNA)of cerebral cavernomas (Notelet et al., 1997 Surg. Neurol. 47: 364-370),a 35 kD tumor-associated autoantigen in papillary thyroid carcinoma(Lucas et al., 1996 Anticancer Res. 16: 2493-2496), CDC27 (including themutated form of the protein), antigens triosephosphate isomerase,707-AP, A60 mycobacterial antigen (Macs et al., 1996, J. Cancer Res.Clin. Oncol. 122: 296-300), Annexin II, AFP, ART-4, BAGE, β-catenin/m,BCL-2, bcr-abl, bcr-abl p190, bcr-abl p210, BRCA-1, BRCA-2, CA 19-9(Tolliver and O′Brien, 1997, South Med. J. 90: 89-90; Tsuruta at al.,1997 Urol. Int. 58: 20-24), CAMEL, CAP-1, CASP-8, CDC27/m, CDK-4/m, CEA(Huang et al., Exper Rev. Vaccines (2002)1:49-63), CT9, CT10, Cyp-B,Dek-cain, DAM-6 (MAGE-B2), DAM-10 (MAGE-B1), EphA2 (Zantek et al., CellGrowth Differ. (1999) 10:629-38; Carles-Kinch et al., Cancer Res. (2002)62:2840-7), EphA4 (Cheng at al., 2002, Cytokine Growth Factor Rev.13:75-85), tumor associated Thomsen-Friedenreich antigen (Dahlenborg etal., 1997, Int. J Cancer 70: 63-71), ELF2M, ETV6-AML1, G250, GAGE-1,GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE-7B, GAGE-8, GnT-V, gp100(Zajac et al., 1997, Int. J Cancer 71: 491-496), HAGE, HER2/neu,HLA-A*0201-R170I, HPV-E7, HSP70-2M, HST-2, hTERT, hTRT, iCE, inhibitorsof apoptosis (e.g., survivin), KH-1 adenocarcinoma antigen (Deshpandeand Danishefsky, 1997, Nature 387: 164-166), KIAA0205, K-ras, LAGE,LAGE-1, LDLR/FUT, MAGE-1, MAGE-2, MAGE-3, MAGE-6, MAGE-A1, MAGE-A2,MAGE-A3, MAGE-A4, MAGE-A6, MAGE-A10, MAGE-Al2, MAGE-B5, MAGE-B6,MAGE-C2, MAGE-C3, MAGE D, MART-1, MART-1/Melan-A (Kawakami andRosenberg, 1997, Int. Rev. Immunol. 14: 173-192), MC1R, MDM-2, Myosin/m,MUC1, MUC2, MUM-1, MUM-2, MUM-3, neo-polyA polymerase, NA88-A, NY-ESO-1,NY-ESO-1a (CAG-3), PAGE-4, PAP, Proteinase 3 (Molldrem et al., Blood(1996) 88:2450-7; Molldrem et al., Blood (1997) 90:2529-34), P15, p190,Pm1/RARα, PRAME, PSA, PSM, PSMA, RAGE, RAS, RCAS1, RU1, RU2, SAGE,SART-1, SART-2, SART-3, SP17, SPAS-1, TEL/AML1, TPI/m, Tyrosinase, TARP,TRP-1 (gp75), TRP-2, TRP-2/INT2, WT-1, and alternatively translatedNY-ESO-ORF2 and CAMEL proteins, derived from the NY-ESO-1 and LAGE-1genes. Numerous other cancer antigens are well known in the art.

Preferably, the cancer/tumor antigen or the cancer/tumor epitope is arecombinant cancer/tumor antigen or a recombinant cancer/tumor epitope.Such a recombinant cancer/tumor antigen or a recombinant cancer/tumorepitope may be designed by introducing mutations that change (add,delete or substitute) particular amino acids in the overall amino acidsequence of the native cancer/tumor antigen or the native cancer/tumorepitope. The introduction of mutations does not alter the cancer/tumorantigen or the cancer/tumor epitope so much that it cannot beuniversally applied across a mammalian subject, and preferably a humanor dog subject, but changes it enough that the resulting amino acidsequence breaks tolerance or is considered a foreign antigen in order togenerate an immune response. Another manner may be creating a consensusrecombinant cancer/tumor antigen or cancer/tumor epitope that has atleast 85% and up to 99% amino acid sequence identity to its'corresponding native cancer/tumor antigen or native cancer/tumorepitope; preferably at least 90% and up to 98% sequence identity; morepreferably at least 93% and up to 98% sequence identity; or even morepreferably at least 95% and up to 98% sequence identity. In someinstances the recombinant cancer/tumor antigen or the recombinantcancer/tumor epitope has 95%, 96%, 97%, 98%, or 99% amino acid sequenceidentity to its' corresponding native cancer/tumor antigen orcancer/tumor epitope. The native cancer/tumor antigen is the antigennormally associated with the particular cancer or cancer tumor.Depending upon the cancer/tumor antigen, the consensus sequence of thecancer/tumor antigen can be across mammalian species or within subtypesof a species or across viral strains or serotypes. Some cancer/tumorantigen do not vary greatly from the wild type amino acid sequence ofthe cancer/tumor antigen. The aforementioned approaches can be combinedso that the final recombinant cancer/tumor antigen or cancer/tumorepitope has a percent similarity to native cancer antigen amino acidsequence as discussed above. Preferably, however, the amino acidsequence of an epitope of a cancer/tumor antigen as described herein isnot mutated and, thus, identical to the reference epitope sequence.

As used herein “pathogen epitope” means an epitope from an antigenicprotein, an antigenic polysaccharide, an antigenic lipid, an antigeniclipoprotein or an antigenic glycolipid from a pathogen includingviruses, bacteria, fungi, protozoa and multicellular parasites.Antigenic proteins, polysaccharides, lipids, lipoproteins or glycolipidsfrom pathogens include, herewith, proteins, polysaccharides, lipids,lipoproteins and glycolipids, respectively, from pathogens responsibleof diseases which can be a target for vaccination including, forinstance, Amoebiasis, Anthrax, Buruli Ulcer (Mycobacterium ulcerans),Caliciviruses associated diarrhoea, Campylobacter diarrhoea, CervicalCancer (Human papillomavirus), Chlamydia trachomatis associated genitaldiseases, Cholera, Crimean-Congo haemorrhagic fever, Dengue Fever,Diptheria, Ebola haemorrhagic fever, Enterotoxigenic Escherichia coli(ETEC) diarrhoea, Gastric Cancer (Helicobacter pylori), Gonorrhea, GroupA Streptococcus associated diseases, Group B Streptococcus associateddiseases, Haemophilus influenzae B pneumonia and invasive disease,Hepatitis A, Hepatitis B, Hepatitis C, Hepatitis E diarrhoea, Herpessimplex type 2 genital ulcers, HIV/AIDS, Hookworm Disease, Influenza,Japanese encephalitis, Lassa Fever, Leishmaniasis, Leptospirosi, Livercancer (Hepatitis B), Liver Cancer (Hepatitis C), Lyme Disease, Malaria,Marburg haemorrhagic fever, Measles, Mumps, Nasopharyngeal cancer(Epstein-Barr virus), Neisseria meningitidis Meningitis, Parainfluenzaassociated pneumonia, Pertussis, Plague, Poliomyelitis, Rabies,Respiratory syncytial virus (RSV) pneumonia, Rift Valley fever,Rotavirus diarrhoea, Rubella, Schistosomiasis, Severe Acute RespiratorySyndrome (SARS), Shigellosis, Smallpox, Staphylococcus aureus associateddiseases, Stomach Cancer (Helicobacter pylori), Streptococcus pneumoniaeand invasive disease, Tetanus, Tick-borne encephalitis, Trachoma,Tuberculosis, Tularaemia, Typhoid fever, West-Nile virus associateddisease, Yellow fever.

Preferably, the at least one antigen or antigenic epitope will bepresented at the cell surface in an MHC class I and/or MHC class IIcontext and/or in a CD1 context, whereby presentation at the cellsurface in an MHC class I and/or MHC class II context is preferred. Thephrase “epitope presentation in the MHC class I context” refers inparticular to a CD8⁺ epitope lying in the groove of a MHC class Imolecule at the surface of a cell. The phrase “epitope presentation inthe MHC class II context” refers in particular to a CD4⁺ epitope lyingin the groove of a MHC class II molecule at the surface of a cell. Thephrase “epitope presentation in the CD1 context” refers in particular toa lipidic epitope lying in the groove of a cluster of differentiation 1molecule at the surface of a cell.

Advantageously, the complex according to the invention comprises a cellpenetrating peptide and at least one antigen or antigenic epitope, andallows the transport and presentation of said epitopes at the cellsurface of antigen presenting cells in an MHC class I and MHC class IIcontext, and is, thus, useful in vaccination and immunotherapy.

Preferably, the complex according to the present invention comprises atleast one antigen or antigenic epitope, which is at least one CD4⁺epitope and/or at least one CD8⁺ epitope.

The terms “CD4⁺ epitope” or “CD4⁺-restricted epitope”, as used herein,designate an epitope recognized by a CD4⁺ T cell, said epitope inparticular consisting of an antigen fragment lying in the groove of aMHC class II molecule. A single CD4⁺ epitope comprised in the complexaccording to the present invention preferably consists of about 12-25amino acids. It can also consist of, for example, about 8-25 amino acidsor about 6-100 amino acids.

The terms “CD8⁺ epitope” or “CD8+-restricted epitope”, as used herein,designate an epitope recognized by a CD8⁺ T cell, said epitope inparticular consisting of an antigen fragment lying in the groove of aMHC class I molecule. A single CD8⁺ epitope comprised in the complexaccording to the present invention preferably consists of about 8-11amino acids. It can also consist of, for example, about 8-15 amino acidsor about 6-100 amino acids.

Preferably, the at least one antigen can comprise or the at least oneantigenic epitope can consist of a CD4⁺ epitope and/or a CD8⁺ epitopecorresponding to antigenic determinant(s) of a cancer/tumor-associatedantigen, a cancer/tumor-specific antigen, or an antigenic protein from apathogen. More preferably, the at least one antigen can comprise or theat least one antigenic epitope can consist of a CD4⁺ epitope and/or aCD8⁺ epitope corresponding to antigenic determinant(s) of acancer/tumor-associated antigen or a cancer/tumor-specific antigen. Mostpreferably, the at least one antigen can comprise or the at least oneantigenic epitope can consist of a CD4⁺ epitope and/or a CD8⁺ epitopecorresponding to antigenic determinant(s) of a tumor-associated antigenor a tumor-specific antigen.

It is also preferred that the complex according to the present inventioncomprises at least two antigens or antigenic epitopes, wherein at leastone antigen or antigenic epitope comprises or consists a CD4⁺ epitopeand at least one antigen or antigenic epitope comprises or consists aCD8⁺ epitope. It is now established that T_(h) cells (CD4⁺) play acentral role in the anti-tumor immune response both in DC licensing andin the recruitment and maintenance of CTLs (CD8⁺) at the tumor site.Therefore, a complex according to the present invention comprising atleast two antigens or antigenic epitopes, wherein at least one antigenor antigenic epitope comprises or consists of a CD4⁺ epitope and atleast one antigen or antigenic epitope comprises or consists a CD8⁺epitope, provides an integrated immune response allowing simultaneouspriming of CTLs and Th cells and is thus preferable to immunity againstonly one CD8⁺ epitope or only one CD4⁺ epitope. For example, the complexaccording to the present invention may preferably comprise anEalpha-CD4⁺ epitope and a gp100-CD8+ epitope.

Preferably, the complex according to the present invention comprises atleast two antigens or antigenic epitopes, wherein the at least twoantigens or antigenic epitopes comprise or consist of at least two, e.g.2, 3, 4, 5, 6, 7, 8, 9, or more, CD4⁺ epitopes and/or at least two, e.g.2, 3, 4, 5, 6, 7, 8, 9, or more, CD8⁺ epitopes. Thereby, the at leasttwo antigens or antigenic epitopes are preferably different antigens orantigenic epitopes, more preferably the at least two antigens orantigenic epitopes are different from each other but relating to thesame kind of tumor. A multi-antigenic vaccine will (i) avoid outgrowthof antigen-loss variants, (ii) target different tumor cells within aheterogeneous tumor mass and (iii) circumvent patient-to-patient tumorvariability. Thus, the complex according to the present inventionparticularly preferably comprises at least four antigens or antigenicepitopes, in particular with at least two CD8⁺ epitopes and at least twoCD4⁺ epitopes. Such a complex according to the present invention inducesmulti-epitopic CD8 CTLs and CD4 T_(h) cells to function synergisticallyto counter tumor cells and promote efficient anti-tumor immunity. T_(h)cells are also involved in the maintenance of long-lasting cellularimmunity that was monitored after vaccination. Such a complex accordingto the present invention induces polyclonal, multi-epitopic immuneresponses and poly-functional CD8⁺ and CD4⁺ T cells, and thusefficacious anti-tumor activity.

Preferably, the complex according to the present invention comprises atleast two antigens or antigenic epitopes, more preferably the complexaccording to the present invention comprises at least three antigens orantigenic epitopes, even more preferably the complex according to thepresent invention comprises at least four antigens or antigenicepitopes, particularly preferably the complex according to the presentinvention comprises at least five antigens or antigenic epitopes andmost preferably the complex according to the present invention comprisesat least six antigens or antigenic epitopes. The antigens or antigenicepitopes comprised by the complex according to the present invention maybe the same or different, preferably the antigens or antigenic epitopescomprised by the complex according to the present invention aredifferent from each other. Preferably, the complex according to thepresent invention comprises at least one CD4⁺ epitope and at least oneCD8⁺ epitope.

Preferably, the complex according to the present invention comprisesmore than one CD4⁺ epitope, e.g. two or more CD4⁺ epitopes from the sameantigen or from different antigens, and preferably no CD8⁺ epitope. Itis also preferred that the complex according to the present inventioncomprises more than one CD8⁺ epitope, e.g. two or more CD8⁺ epitopesfrom the same antigen or from different antigens, and preferably no CD4⁺epitope. Most preferably, however, the complex according to the presentinvention comprises (i) at least one CD4⁺ epitope, e.g. two or more CD4⁺epitopes from the same antigen or from different antigens, and (ii) atleast one CD8⁺ epitope, e.g. two or more CD8⁺ epitopes from the sameantigen or from different antigens.

For example, the complex according to the present invention maypreferably comprise a gp100-CD8⁺ epitope, an Ealpha-CD4⁺ epitope, and afurther CD4⁺ epitope and a further CD8⁺ epitope. Even more preferably,the complex according to the present invention may comprise apolypeptide or protein comprising a gp100-CD8⁺ epitope and anEalpha-CD4⁺ epitope. For example, such a polypeptide or proteincomprised by the complex according to the present invention comprises orconsists of an amino acid sequence according to SEQ ID NO: 14 orsequence variants thereof as defined above:

ESLKIS QAVHAAHAEI NEAGREVVGV GALKVPRNQD WLGVPRFAKF ASFEAQGALA NIAVDKANLDVEQLESIINF EKLTEWTGS

SEQ ID NO: 14 (MADS-cargo comprising OVA-CD4⁺, gp100-CD8⁺, Ealpha-CD4⁺,and OVA-CD8⁺ epitopes)

For example, the complex according to the present invention may alsocomprise a gp70-CD8⁺ epitope and/or a gp70-CD4⁺ epitope. In particular,the complex according to the present invention may comprise apolypeptide or protein comprising a gp70-CD8⁺ epitope and/or a gp70-CD4⁺epitope. For example, such a polypeptide or protein comprised by thecomplex according to the present invention comprises or consists of anamino acid sequence according to SEQ ID NO: 43 or sequence variantsthereof as defined above:

VTYHSPSYAYHQFERRAILNRLVQFIKDRI

SEQ ID NO: 43 (Mad8-cargo comprising a gp70-CD8⁺ and a gp70-CD4⁺epitope)

For example, the complex according to the present invention maypreferably comprise at least one survivin epitope, such as a survivinCD8⁺ epitope and/or a survivin CD4⁺ epitope. More preferably, thecomplex according to the present invention may comprise a polypeptide orprotein comprising a survivin CD8⁺ epitope and/or a survivin CD4⁺epitope. More preferably, the complex according to the present inventionmay comprise a polypeptide or protein comprising more than one survivinCD8⁺ epitope and/or more than one survivin CD4⁺ epitope, such as twodifferent survivin CD8⁺ epitopes. For example, such a polypeptide orprotein comprised by the complex according to the present inventioncomprises or consists of an amino acid sequence according to SEQ ID NO:44 or sequence variants thereof as defined above:

NYRIATFKNWPFLEDCAMEELTVSEFLKLDRQR

SEQ ID NO: 44 (Mad11-cargo comprising survivin CD8⁺ epitope 1 andsurvivin CD8⁺ epitope 2)

For example, the complex according to the present invention maypreferably comprise an epitope from a neoantigen. Even more preferably,the complex according to the present invention may comprise apolypeptide or protein comprising an epitope from a neoantigen, such asthe neoantigen from MC-38 tumor cell line identified by Yadav et al.Nature. 2014 Nov. 27; 515(7528):572-6. For example, such a polypeptideor protein comprised by the complex according to the present inventioncomprises or consists of an amino acid sequence according to SEQ ID NO:42 or sequence variants thereof as defined above:

HLELASMTNMELMSSIV

SEQ ID NO: 42 (Mad9-cargo comprising the epitope from a neoantigen asdescribed by Yadav et al. Nature. 2014 Nov. 27; 515(7528):572-6).

For example, the complex according to the present invention maypreferably comprise more than one, e.g. two or three, epitopes fromneoantigens. Even more preferably, the complex according to the presentinvention may comprise a polypeptide or protein comprising more thanone, e.g. two or three, epitopes from neoantigens, such as theneoantigens from MC-38 tumor cell line identified by Yadav et al.Nature. 2014 Nov. 27; 515(7528):572-6. For example, such a polypeptideor protein comprised by the complex according to the present inventioncomprises or consists of an amino acid sequence according to SEQ ID NO:63 or sequence variants thereof as defined above:

LFRAAQLANDVVLQIMEHLELASMTNMELMSSIVVISASIIVFNLLELEG

SEQ ID NO: 63 (Mad12-cargo comprising the epitope from a neoantigen asdescribed by Yadav et al. Nature. 2014 Nov. 27; 515(7528):572-6).

Preferably, the at least one antigen or antigenic epitope comprised bythe complex according to the present invention is a peptide,polypeptide, or a protein. Examples of antigen or antigenic epitope ofpeptidic, polypeptidic, or proteic nature useful in the invention,include cancer/tumor antigens or antigenic epitopes thereof, allergyantigens or antigenic epitopes thereof, auto-immune self-antigens orantigenic epitopes thereof, pathogenic antigens or antigenic epitopesthereof, and antigens or antigenic epitopes thereof from viruses,preferably from cytomegalovirus (CMV), orthopox variola virus, orthopoxalastrim virus, parapox ovis virus, molluscum contagiosum virus, herpessimplex virus 1, herpes simplex virus 2, herpes B virus, varicellazoster virus, pseudorabies virus, human cytomegaly virus, human herpesvirus 6, human herpes virus 7, Epstein-Barr virus, human herpes virus 8,hepatitis B virus, chikungunya virus, O′nyong'nyong virus, rubivirus,hepatitis C virus, GB virus C, West Nile virus, dengue virus, yellowfever virus, louping ill virus, St. Louis encephalitis virus, Japan Bencephalitis virus, Powassan virus, FSME virus, SARS, SARS-associatedcorona virus, human corona virus 229E, human corona virus Oc43,Torovirus, human T cell lymphotropic virus type I, human T celllymphotropic virus type II, HIV (AIDS), i.e. human immunodeficiencyvirus type 1 or human immunodeficiency virus type 2, influenza virus,Lassa virus, lymphocytic choriomeningitis virus, Tacaribe virus, Juninvirus, Machupo virus, Borna disease virus, Bunyamwera virus, Californiaencephalitis virus, Rift Valley fever virus, sand fly fever virus,Toscana virus, Crimean-Congo haemorrhagic fever virus, Hazara virus,Khasan virus, Hantaan virus, Seoul virus, Prospect Hill virus, Puumalavirus, Dobrava Belgrade virus, Tula virus, sin nombre virus, LakeVictoria Marburg virus, Zaire Ebola virus, Sudan Ebola virus, IvoryCoast Ebola virus, influenza virus A, influenza virus B, influenzaviruses C, parainfluenza virus, malaria parasite (Plasmodium falciparum,Plasmodium vivax, Plasmodium ovale, Plasmodium malariae, Plasmodiumknowlesi), Marburg virus, measles virus, mumps virus, respiratorysyncytial virus, human metapneumovirus, vesicular stomatitis Indianavirus, rabies virus, Mokola virus, Duvenhage virus, European batlyssavirus 1+2, Australian bat lyssavirus, adenoviruses A-F, humanpapilloma viruses, condyloma virus 6, condyloma virus 11, polyomaviruses, adeno-associated virus 2, rotaviruses, orbiviruses, varicellaincluding varizella zoster, etc., or antigens or antigenic epitopes fromleishmania, typanosomes, amibes, bacteria, etc., or may be selected fromepitopes or from variants of the above antigens or antigenic epitopes.Preferably, epitopes as well as variants of antigens as defined aboveexhibit a sequence homology or identity of about 10%, in particular atleast 10%, about 20%, in particular at least 20%, about 30%, inparticular at least 30%, about 40%, in particular at least 40%, about50%, in particular at least 50%, about 60%, in particular at least 60%,about 70%, in particular at least 70%, about 80%, in particular at least80%, about 90% in particular at least 90%, at least 95% or at least 98%with one of the antigens or antigen sequences as shown or describedabove. In this context, the definition of epitopes and variantssimilarly applies as defined.

Examples of antigens or antigenic epitopes in the category of peptide,polypeptide or protein include a combination of multiple glioma epitopessuch as those described in Novellino et al (2005, Cancer ImmunolImmunother, 54(3):187-207), Vigneron et al. (2013, Cancer Immun. 13:15).However, a single complex according to the present invention may alsocomprise only a subset, i.e. one or more of all of said glioma epitopes.In such a case preferably different complexes according to the presentinvention comprise different subsets of all of said glioma epitopes, sothat for example a vaccine according to the present invention comprisingsuch different complexes according to the present invention comprisesall of said glioma epitopes but distributed in the different complexes.

Moreover, a complex according to the invention may also comprise atleast one antigen or antigenic epitope, wherein said antigen orantigenic epitope is a polysaccharide, a lipid, a lipoprotein, and/or aglycolipid, in particular a polysaccharidic, lipidic, lipoproteic,and/or glycolipidic epitope, which can be, for example, pathogenepitopes as defined herewith.

In particular, the complex according to the invention may comprise atleast one antigen or antigenic epitope, wherein said antigen orantigenic epitope is polysaccharidic, lipidic, lipoproteic, and/orglycolipidic, including viral, bacterial, fungal, protozoal andmulticellular parasitic antigens or antigenic epitopes.

Preferably, said epitopes will be presented at the cell surface in anMHC class I and/or MHC class II context.

Preferably, said lipidic epitopes will be presented at the cell surfacein a CD1 (cluster of differentiation 1) context.

The complex according to the present invention may also comprise atleast one antigen or antigenic epitope, wherein said antigen orantigenic epitope is a small molecule drug or toxin.

Examples of cargo molecules within the category of small molecule drugsor toxins useful in the invention include cyclosporine A, paclitaxel,doxorubicin, methotrexate, 5-aminolevulinic acid, diphtheria toxin,sunitinib and those molecules reviewed in De wit Amer (2010, NeuroOncol, 12(3):304-16).

The complex according to the present invention comprises at least oneantigen or antigenic epitope, preferably the complex according to thepresent invention comprises more than one antigen or antigenic epitope,in particular 2, 3, 4, 5, 6, 7, 8, 9, 10 or more antigens or antigenicepitopes, more preferably the complex according to the present inventioncomprises (at least) two or three antigens or antigenic epitopes, evenmore preferably the complex according to the present invention comprises(at least) four or five antigens or antigenic epitopes.

If more than one antigen or antigenic epitope is comprised by thecomplex according to the present invention it is understood that saidantigen or antigenic epitope is in particular also covalently linked inthe complex according to the present invention, e.g. to another antigenor antigenic epitope and/or to a component a), i.e. a cell penetratingpeptide, and/or to a component c), i.e. a TLR peptide agonist.

The various antigens or antigenic epitopes comprised by the complexaccording to the present invention may be the same or different.Preferably, the various antigens or antigenic epitopes comprised by thecomplex according to the present invention are different from eachother, thus providing a multi-antigenic and/or multi-epitopic complex.

Moreover, it is preferred that the more than one antigen or antigenicepitope, in particular 2, 3, 4, 5, 6, 7, 8, 9, 10 or more antigens orantigenic epitopes, are positioned consecutively in the complexaccording to the present invention. This means in particular that allantigens and/or antigenic epitopes comprised by the complex arepositioned in a stretch, which is neither interrupted by component a),i.e. a cell penetrating peptide, nor by component c), i.e. a TLR peptideagonist. Rather, component a) and component c) are positioned in thecomplex for example before or after such a stretch of all antigensand/or antigenic epitopes. However, the antigens and/or antigenicepitopes positioned consecutively in such a way may be linked to eachother for example by a spacer or linker as described below, which isneither component a), i.e. a cell penetrating peptide, nor component c),i.e. a TLR peptide agonist.

Alternatively, however, the various antigens and/or antigenic epitopesmay also be positioned in any other way in the complex according to thepresent invention, for example with component a) and/or component c)positioned in between two or more antigens and/or antigenic epitopes,i.e. with one or more antigens and/or antigenic epitopes positionedbetween component a) and component c) (or vice versa) and, optionally,one or more antigens and/or antigenic epitopes positioned at therespective other end of component a) and/or component c).

It is understood that a number of different antigens or antigenicepitopes relating to the same kind of disease, in particular to the samekind of tumor, may be advantageously comprised by a single complexaccording to the present invention. Alternatively, a number of differentantigens or antigenic epitopes relating to the same kind of disease, inparticular to the same kind of tumor, may be distributed to subsets ofdifferent antigens or antigenic epitopes, in particular subsetscomplementing each other in the context of a certain kind of disease,e.g. tumor, which are comprised by different complexes according to thepresent invention, whereby such different complexes comprising differentsubsets may advantageously be administered simultaneously, e.g. in asingle vaccine, to a subject in need thereof.

Preferably, the complex according to the present invention comprises atleast one tumor epitope, which is an epitope of an antigen selected fromthe group consisting of EpCAM, HER-2/neu, MUC-1, TOMM34, RNF 43, KOC1,VEGFR, βhCG, survivin, CEA, TGFβR2, p53, KRas, OGT, CASP5, COA-1, MAGE,SART, IL13Ralpha2, CMV, EGFRvIII, EphA2, gp100, hTert, TRP-2, YKL-40,brevican, neuroligin 4 and PTPRz1. More preferably, the complexaccording to the present invention comprises at least one tumor antigenselected from the group consisting of EpCAM, HER-2/neu, MUC-1, TOMM34,RNF 43, KOC1, VEGFR, βhCG, survivin, CEA, TGFβR2, p53, KRas, OGT, CASP5,COA-1, MAGE, SART, IL13Ralpha2, CMV, EGFRvIII, EphA2, gp100, hTert,TRP-2, YKL-40, brevican, neuroligin 4 and PTPRz1 or a fragment thereof,or a sequence variant of a tumor antigen or a sequence variant of afragment thereof. It is also preferred that the complex according to thepresent invention comprises at least one tumor epitope, which is anepitope of an antigen selected from the glioma antigens disclosed byReardon, D. A., et al., An update on vaccine therapy and otherimmunotherapeutic approaches for glioblastoma. Expert Rev Vaccines,2013. 12(6): p. 597-615.

As used herein, a “fragment” of an antigen comprises at least 10consecutive amino acids of the antigen, preferably at least 15consecutive amino acids of the antigen, more preferably at least 20consecutive amino acids of the antigen, even more preferably at least 25consecutive amino acids of the antigen and most preferably at least 30consecutive amino acids of the antigen. A “sequence variant” is asdefined above, namely a sequence variant has an (amino acid) sequencewhich is at least 70%, at least 75%, preferably at least 80%, morepreferably at least 85%, even more preferably at least 90%, particularlypreferably at least 95%, most preferably at least 99% identical to thereference sequence. A “functional” sequence variant means in the contextof an antigen/antigen fragment/epitope, that the function of theepitope(s), e.g. comprised by the antigen (fragment), is not impaired orabolished. Preferably, however, the amino acid sequence of theepitope(s), e.g. comprised by the cancer/tumor antigen (fragment) asdescribed herein, is not mutated and, thus, identical to the referenceepitope sequence.

As described above, suitable cancer/tumor epitopes of those antigens areknown from the literature or can be identified by using cancer/tumorepitope databases, e.g. from van der Bruggen P, Stroobant V, Vigneron N,Van den Eynde B. Peptide database: T cell-defined tumor antigens. Cancerlmmun 2013; URL: http://www.cancerimmunity.org/peptide/, wherein humantumor antigens recognized by CD4+ or CD8+ T cells are classified intofour major groups on the basis of their expression pattern, or from thedatabase “Tantigen” (TANTIGEN version 1.0, Dec 1, 2009; developed byBioinformatics Core at Cancer Vaccine Center, Dana-Farber CancerInstitute; URL: http://cvc.dfci.harvard.edu/tadb/).

EpCAM

Ep-Cam is a glycoprotein mediating cellular adhesion. The amino acidsequence of EpCAM is shown in the following:

[SEQ ID NO: 47] MAPPQVLAFGLLLAAATATFAAAQEECVCENYKLAVNCFVNNNRQCQCTSVGAQNTVICSKLAAKCLVMKAEMNGSKLGRRAKPEGALQNNDGLYDPDCDESGLFKAKQCNGTSMCWCVNTAGVRRTDKDTEITCSERVRTYWIIIELKHKAREKPYDSKSLRTALQKEITTRYQLDPKFITSILYENNVITIDLVQNSSQKTQNDVDIADVAYYFEKDVKGESLFHSKKMDLTVNGEQLDLDPGQTLIYYVDEKAPEFSMQGLKAGVIAVIVVVVIAVVAGIVVLVISRKKRMAKYEKA EIKEMGEMHRELNA

Accordingly, a preferred complex according to the present inventioncomprises an amino acid sequence according to SEQ ID NO: 47 or afragment or a variant thereof as described herein.

Several epitopes of EpCAM are known to the skilled person. A preferredEpCAM epitope, which is preferably comprised by the complex according tothe present invention, includes the following epitope (the epitopesequence shown in the following is a fragment of the above EpCAMsequence and is, thus, shown in the above EpCAM sequence underlined; thefollowing epitope sequence may refer to one epitope or more than one(overlapping) epitopes):

[SEQ ID NO: 48] GLKAGVIAV

Accordingly, a preferred complex according to the present inventioncomprises an amino acid sequence according to SEQ ID NO: 48.

HER-2/neu

Her-2 belongs to the EGFR (epidermal growth factor receptor) family.Many HLA-A epitopes are known to the skilled person. The amino acidsequence of HER2 is shown in the following:

[SEQ ID NO: 70] MELAALCRWGLLLALLPPGAASTQVCTGTDMKLRLPASPETHLDMLRHLYQGCQVVQGNLELTYLPTNASLSFLQDIQEVQGYVLIAHNQVRQVPLQRLRIVRGTQLFEDNYALAVLDNGDPLNNTTPVTGASPGGLRELQLRSLTEILKGGVLIQRNPQLCYQDTILWKDIFHKNNQLALTLIDTNRSRACHPCSPMCKGSRCWGESSEDCQSLTRTVCAGGCARCKGPLPTDCCHEQCAAGCTGPKHSDCLACLHFNHSGICELHCPALVTYNTDTFESMPNPEGRYTFGASCVTACPYNYLSTDVGSCTLVCPLHNQEVTAEDGTQRCEKCSKPCARVCYGLGMEHLREVRAVTSANIQEFAGCKKIFGSLAFLPESFDGDPASNTAPLQPEQLQVFETLEEITGYLYISAWPDSLPDLSVFQNLQVIRGRILHNGAYSLTLQGLGISWLGLRSLRELGSGLALIHHNTHLCFVHTVPWDQLFRNPHQALLHTANRPEDECVGEGLACHQLCARGHCWGPGPTQCVNCSQFLRGQECVEECRVLQGLPREYVNARHCLPCHPECQPQNGSVTCFGPEADQCVACAHYKDPPFCVARCPSGVKPDLSYMPIWKFPDEEGACQPCPINCTHSCVDLDDKGCPAEQRASPLTSIISAVVGILLVVVLGVVFGILIKRRQQKIRKYTMRRLLQETELVEPLTPSGAMPNQAQMRILKETELRKVKVLGSGAFGTVYKGIWIPDGENVKIPVAIKVLRENTSPKANKEILDEAYVMAGVGSPYVSRLLGICLTSTVQLVTQLMPYGCLLDHVRENRGRLGSQDLLNWCMQIAKGMSYLEDVRLVHRDLAARNVLVKSPNHVKITDFGLARLLDIDETEYHADGGKVPIKWMALESILRRRFTHQSDVWSYGVTVWELMTFGAKPYDGIPAREIPDLLEKGERLPQPPICTIDVYMIMVKCWMIDSECRPRFRELVSEFSRMARDPQRFVVIQNEDLGPASPLDSTFYRSLLEDDDMGDLVDAEEYLVPQQGFFCPDPAPGAGGMVHHRHRSSSTRSGGGDLTLGLEPSEEEAPRSPLAPSEGAGSDVFDGDLGMGAAKGLQSLPTHDPSPLQRYSEDPTVPLPSETDGYVAPLTCSPQPEYVNQPDVRPQPPSPREGPLPAARPAGATLERPKTLSPGKNGVVKDVFAFGGAVENPEYLTPQGGAAPQPHPPPAFSPAFDNLYYWDQDPPERGAPPSTFKGTPTAENPEYLG LDVPV

Accordingly, a preferred complex according to the present inventioncomprises an amino acid sequence according to SEQ ID NO: 70 or afragment or a variant thereof as described herein. As described above,suitable cancer/tumor epitopes of Her-2 are known from the literature orcan be identified by using cancer/tumor epitope databases, e.g. from vander Bruggen P, Stroobant V, Vigneron N, Van den Eynde B. Peptidedatabase: T cell-defined tumor antigens. Cancer Immun 2013; URL:http://www.cancerimmunity.org/peptide/, wherein human tumor antigensrecognized by CD4+ or CD8+ T cells are classified into four major groupson the basis of their expression pattern, or from the database“Tantigen” (TANTIGEN version 1.0, Dec 1, 2009; developed byBioinformatics Core at Cancer Vaccine Center, Dana-Farber CancerInstitute; URL: http://cvc.dfci.harvard.edu/tadb/).

Mucin-1 (MUC-1)

MUC-1 is a human epithelial mucin, acting on cell adhesion. The aminoacid sequence of MUC-1 is shown in the following:

[SEQ ID NO: 49] MTPGTQSPFFLLLLLTVLTVVTGSGHASSTPGGEKETSATQRSSVPSSTEKNAVSMTSSVLSSHSPGSGSSTTQGQDVTLAPATEPASGSAATWGQDVTSVPVTRPALGSTTPPAHDVTSAPDNKPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDNRPALGSTAPPVHNVTSASGSASGSASTLVHNGTSARATTTPASKSTPFSIPSHHSDTPTTLASHSTKTDASSTHHSSVPPLTSSNHSTSPQLSTGVSFFFLSFHISNLQFNSSLEDPSTDYYQELQRDISEMFLQIYKQGGFLGLSNIKFRPGSVVVQLTLAFREGTINVHDVETQFNQYKTEAASRYNLTISDVSVSDVPFPFSAQSGAGVPGWGIALLVLVCVLVALAIVYLIALAVCQCRRKNYGQLDIFPARDTYHPMSEYPTYHTHGRYVPPSSTDRSPYEKVSAGNGGSSLSYTNPAVAA TSANL

Accordingly, a preferred complex according to the present inventioncomprises an amino acid sequence according to SEQ ID NO: 49 or afragment or a variant thereof as described herein.

Several epitopes of MUC-1 are known to the skilled person. PreferredMUC-1 epitopes, which are preferably comprised by the complex accordingto the present invention, include the following epitopes (the epitopesequences shown in the following are fragments of the above MUC-1sequence and are, thus, shown in the above MUC-1 sequence underlined;each of the following epitope sequences may refer to one epitope or morethan one (overlapping) epitopes):

[SEQ ID NO: 50] GSTAPPVHN [SEQ ID NO: 51] TAPPAHGVTS

Accordingly, a preferred complex according to the present inventioncomprises an amino acid sequence according to SEQ ID NO: 50 and/or anamino acid sequence according to SEQ ID NO: 51.

TOMM34

TOMM34 is involved in the import of precursor proteins intomitochondria. Many epitopes thereof are known to the skilled person.

RNF 43

RNF43 is a RING-type E3 ubiquitin ligase and is predicted to contain atransmembrane domain, a protease-associated domain, an ectodomain, and acytoplasmic RING domain. RNF43 is thought to negatively regulate Wntsignaling, and expression of RNF43 results in an increase inubiquitination of frizzled receptors, an alteration in their subcellulardistribution, resulting in reduced surface levels of these receptors.Many epitopes thereof are known to the skilled person.

KOC1

KOC1, also known as insulin-like growth factor 2 mRNA-binding protein 3(IGF2BP3) is an mRNA binding protein. No expression data are howeveravailable.

Vascular Endothelial Growth Factor (VEGF)/Vascular Endothelial GrowthFactor Receptor (VEGFR)

Vascular endothelial growth factor (VEGF), originally known as vascularpermeability factor (VPF), is a signal protein produced by cells thatstimulates vasculogenesis and angiogenesis. It is part of the systemthat restores the oxygen supply to tissues when blood circulation isinadequate. VEGF's normal function is to create new blood vessels duringembryonic development, new blood vessels after injury, muscle followingexercise, and new vessels (collateral circulation) to bypass blockedvessels. There are three main subtypes of the receptors for VEGF(VEGFR), namely VEGFR1, VEGFR2 and VEGFR3.

Beta Subunit of Human Chorionic Gonadotropin (βhCG)

Human chorionic gonadotropin (hCG) is a hormone produced by the embryofollowing implantation. Some cancerous tumors produce this hormone;therefore, elevated levels measured when the patient is not pregnant canlead to a cancer diagnosis. hCG is heterodimeric with an a (alpha)subunit identical to that of luteinizing hormone (LH),follicle-stimulating hormone (FSH), thyroid-stimulating hormone (TSH),and β (beta) subunit that is unique to hCG. The β-subunit of hCGgonadotropin (beta-hCG) contains 145 amino acids and is encoded by sixhighly homologous genes.

Survivin

Survivin, also called baculoviral inhibitor of apoptosisrepeat-containing 5 or BIRC5, is a member of the inhibitor of apoptosis(IAP) family. The survivin protein functions to inhibit caspaseactivation, thereby leading to negative regulation of apoptosis orprogrammed cell death. The amino acid sequence of survivin is shown inthe following:

[SEQ ID NO: 52] MGAPTLPPAWQPFLKDHRISTFKNWPFLEGCACTPERMAEAGFIHCPTENEPDLAQCFFCFKELEGWEPDDDPIEEHKKHSSGCAFLSVKKQFEELTLGEFLKLDRERAKNKIAKETNNKKKEFEETAKKVRRAIEQLAAMD

Accordingly, a preferred complex according to the present inventioncomprises an amino acid sequence according to SEQ ID NO: 52 or afragment or a variant thereof as described herein.

Several epitopes of survivin are known to the skilled person. Apreferred survivin epitope, which is preferably comprised by the complexaccording to the present invention, includes the following epitope (theepitope sequence shown in the following is a fragment of the abovesurvivin sequence and is, thus, shown in the above survivin sequenceunderlined; the following epitope sequence may refer to one epitope ormore than one (overlapping) epitopes):

[SEQ ID NO: 53] RISTFKNWPF

Accordingly, a preferred complex according to the present inventioncomprises an amino acid sequence according to SEQ ID NO: 53.

Carcino-Embryonic Antigen (CEA)

CEA is an intracellular adhesion glycoprotein. CEA is normally producedin gastrointestinal tissue during fetal development, but the productionstops before birth. Therefore, CEA is usually present only at very lowlevels in the blood of healthy adults. The amino acid sequence of CEA isshown in the following:

[SEQ ID NO: 54] MESPSAPPHRWCIPWQRLLLTASLLTFWNPPTTAKLTIESTPFNVAEGKEVLLLVHNLPQHLFGYSWYKGERVDGNRQIIGYVIGTQQATPGPAYSGREIIYPNASLLIQNIIQNDTGFYTLHVIKSDLVNEEATGQFRVYPELPKPSISSNNSKPVEDKDAVAFTCEPETQDATYLWWVNNQSLPVSPRLQLSNGNRTLTLFNVTRNDTASYKCETQNPVSARRSDSVILNVLYGPDAPTISPLNTSYRSGENLNLSCHAASNPPAQYSWFVNGTFQQSTQELFIPNITVNNSGSYTCQAHNSDTGLNRTTVTTITVYAEPPKPFITSNNSNPVEDEDAVALTCEPEIQNTTYLWWVNNQSLPVSPRLQLSNDNRTLTLLSVTRNDVGPYECGIQNKLSVDHSDPVILNVLYGPDDPTISPSYTYYRPGVNLSLSCHAASNPPAQYSWLIDGNIQQHTQELFISNITEKNSGLYTCQANNSASGHSRTTVKTITVSAELPKPSISSNNSKPVEDKDAVAFTCEPEAQNTTYLWWVNGQSLPVSPRLQLSNGNRTLTLFNVTRNDARAYVCGIQNSVSANRSDPVTLDVLYGPDTPIISPPDSSYLSGANLNLSCHSASNPSPQYSWRINGIPQQHTQVLFIAKITPNNNGTYACFVSNLATGRNNSIVKSITVSASGTSPGLSAGATVGIMIGVLVGVA L

Accordingly, a preferred complex according to the present inventioncomprises an amino acid sequence according to SEQ ID NO: 54 or afragment or a variant thereof as described herein.

Several epitopes of CEA are known to the skilled person. Preferred CEAepitopes, which are preferably comprised by the complex according to thepresent invention, include the following epitopes (the epitope sequencesshown in the following are fragments of the above CEA sequence and are,thus, shown in the above CEA sequence underlined; each of the followingepitope sequences may refer to one epitope or more than one(overlapping) epitopes):

[SEQ ID NO: 55] YLSGANLNLS [SEQ ID NO: 56] SWRINGIPQQ

Accordingly, a preferred complex according to the present inventioncomprises an amino acid sequence according to SEQ ID NO: 55 and/or anamino acid sequence according to SEQ ID NO: 56.

Transforming Growth Factor Beta Receptor 2 (TGFβR2)

TGFβ receptors are single pass serine/threonine kinase receptors. Theyexist in several different isoforms. TGFβR2 is a transmembrane proteinthat has a protein kinase domain, forms a heterodimeric complex withanother receptor protein, and binds TGF-beta. This receptor/ligandcomplex phosphorylates proteins, which then enter the nucleus andregulate the transcription of a subset of genes related to cellproliferation.

P53

P53 is a tumor suppressor protein having a role in preventing genomemutation. P53 has many mechanisms of anticancer function and plays arole in apoptosis, genomic stability, and inhibition of angiogenesis. Inits anti-cancer role, p53 works through several mechanisms: it anactivate DNA repair proteins when DNA has sustained damage; it canarrest growth by holding the cell cycle at the G1/S regulation point onDNA damage recognition; and it can initiate apoptosis.

Kirsten Ras (KRas)

GTPase KRas also known as V-Ki-ras2 Kirsten rat sarcoma viral oncogenehomolog and KRAS, performs an essential function in normal tissuesignaling, and the mutation of a KRAS gene is an essential step in thedevelopment of many cancers. Like other members of the ras subfamily,the KRAS protein is a GTPase and is an early player in many signaltransduction pathways. KRAS is usually tethered to cell membranesbecause of the presence of an isoprene group on its C-terminus. Theamino acid sequence of KRas is shown in the following:

[SEQ ID NO: 57] MTEYKLVVVGAGGVGKSALTIQLIQNHFVDEYDPTIEDSYRKQVVIDGETCLLDILDTAGQEEYSAMRDQYMRTGEGFLCVFAINNTKSFEDIHHYREQIKRVKDSEDVPMVLVGNKCDLPSRTVDTKQAQDLARSYGIPFIETSAKTRQRVEDAFYTLVREIRQYRLKKISKEEKTPGCVKIKKCIIM

Accordingly, a preferred complex according to the present inventioncomprises an amino acid sequence according to SEQ ID NO: 57 or afragment or a variant thereof as described herein.

Several epitopes of Kirsten Ras are known to the skilled person. Apreferred Kirsten Ras epitope, which is preferably comprised by thecomplex according to the present invention, includes the followingepitope (the epitope sequence shown in the following is a fragment ofthe above Kirsten Ras sequence and is, thus, shown in the above KirstenRas sequence underlined; the following epitope sequence may refer to oneepitope or more than one (overlapping) epitopes):

[SEQ ID NO: 58] VVVGAGGVG

Accordingly, a preferred complex according to the present inventioncomprises an amino acid sequence according to SEQ ID NO: 58.

O-Linked N-Acetylglucosamine (GlcNAc) Transferase (OGT)

OGT (O-Linked N-Acetylglucosamine (GlcNAc) Transferase, O-GlcNActransferase, OGTase, O-linked N-acetylglucosaminyltransferase, uridinediphospho-N-acetylglucosamine:polypeptidebeta-N-acetylglucosaminyltransferase, protein O-linkedbeta-N-acetylglucosamine transferase) is an enzyme with system nameUDP-N-acetyl-D-glucosamine:protein-O-beta-N-acetyl-D-glucosaminyltransferase) is an enzyme with system name “UDP-N-acetyl-D-glucosamine:protein-O-beta-N-acetyl-D-glucosaminyl transferase”. OGT catalyzesthe addition of a single N-acetylglucosamine in O-glycosidic linkage toserine or threonine residues of intracellular proteins. OGT is a part ofa host of biological functions within the human body. OGT is involved inthe resistance of insulin in muscle cells and adipocytes by inhibitingthe Threonine 308 phosphorylation of AKT1, increasing the rate of IRS1phosphorylation (at Serine 307 and Serine 632/635), reducing insulinsignaling, and glycosylating components of insulin signals.Additionally, OGT catalyzes intracellular glycosylation of serine andthreonine residues with the addition of N-acetylglucosamine. Studiesshow that OGT alleles are vital for embryogenesis, and that OGT isnecessary for intracellular glycosylation and embryonic stem cellvitality. OGT also catalyzes the posttranslational modification thatmodifies transcription factors and RNA polymerase II, however thespecific function of this modification is mostly unknown.

Caspase 5 (CASP5)

Caspase 5 is an enzyme that proteolytically cleaves other proteins at anaspartic acid residue, and belongs to a family of cysteine proteasescalled caspases. It is an inflammatory caspase, along with caspase 1,caspase 4 and the murine caspase 4 homolog caspase 11, and has a role inthe immune system.

Colorectal Tumor-Associated Antigen-1 (COA-1)

COA-1 was identified in 2003 by Maccalli et al. (Maccalli, C., et al.,Identification of a colorectal tumor-associated antigen (COA-1)recognized by CD4(+) T lymphocytes. Cancer Res, 2003. 63(20): p.6735-43) as strongly expressed by colorectal and melanoma cells (no dataavailable). Its mutation may interfere with the differential recognitionof tumor and normal cells.

Melanoma-Associated Antigen (MAGE)

The mammalian members of the MAGE (melanoma-associated antigen) genefamily were originally described as completely silent in normal adulttissues, with the exception of male germ cells and, for some of them,placenta. By contrast, these genes were expressed in various kinds oftumors. Therefore, the complex according to the present inventionpreferably comprises an antigen of the MAGE-family (a “MAGE” antigen) oran epitope thereof. Of the MAGE family, in particular MAGE-A3 andMAGE-D4 are preferred, and MAGE-A3 is particularly preferred. The normalfunction of MAGE-A3 in healthy cells is unknown. MAGE-A3 is atumor-specific protein, and has been identified on many tumors. Theamino acid sequence of MAGE-A3 is shown in the following:

[SEQ ID NO: 59] MPLEQRSQHCKPEEGLEARGEALGLVGAQAPATEEQEAASSSSTLVEVTLGEVPAAESPDPPQSPQGASSLPTTMNYPLWSQSYEDSSNQEEEGPSTFPDLESEFQAALSRKVAELVHFLLLKYRAREPVTKAEMLGSVVGNWQYFFPVIFSKAFSSLQLVFGIELMEVDPIGHLYIFATCLGLSYDGLLGDNQIMPKAGLLIIVLAIIAREGDCAPEEKIWEELSVLEVFEGREDSILGDPKKLLTQHFVQENYLEYRQVPGSDPACYEFLWGPRALVETSYVKVLHHMVKISGGPHIS YPPLHEWVLREGEE

Accordingly, a preferred complex according to the present inventioncomprises an amino acid sequence according to SEQ ID NO: 59 or afragment or a variant thereof as described herein.

Several epitopes of MAGE-A3 are known to the skilled person. A preferredMAGE-A3 epitope, which is preferably comprised by the complex accordingto the present invention, includes the following epitope (the epitopesequence shown in the following is a fragment of the above MAGE-A3sequence and is, thus, shown in the above MAGE-A3 sequence underlined;the following epitope sequence may refer to one epitope or more than one(overlapping) epitopes):

KVAELVHFL

[SEQ ID NO: 60]

Accordingly, a preferred complex according to the present inventioncomprises an amino acid sequence according to SEQ ID NO: 60.

Squamous Cell Carcinoma Antigen Recognized by T-Cells (SART)

Within the SART family, SART-3 is most preferred. Thus, the complexaccording to the present invention preferably comprises an antigen ofthe SART-family (a “SART” antigen) or an epitope thereof; the complexaccording to the present invention more preferably comprises SART-3 oran epitope thereof. Squamous cell carcinoma antigen recognized byT-cells 3 possesses tumor epitopes capable of inducingHLA-A24-restricted and tumor-specific cytotoxic T lymphocytes in cancerpatients. SART-3 is thought to be involved in the regulation of mRNAsplicing.

IL13Ralpha2

IL13Ralpha2 binds interleukin 13 (IL-13) with very high affinity (andcan therefore sequester it) but does not allow IL-4 binding. It acts asa negative regulator of both IL-13 and IL-4, however the mechanism ofthis is still undetermined. The amino acid sequence of IL13Ralpha2 isshown in the following:

[SEQ ID NO: 61] MAFVCLAIGCLYTFLISTTFGCTSSSDTEIKVNPPQDFEIVDPGYLGYLYLQWQPPLSLDHFKECTVEYELKYRNIGSETWKTIITKNLHYKDGFDLNKGIEAKIHTLLPWQCTNGSEVQSSWAETTYWISPQGIPETKVQDMDCVYYNWQYLLCSWKPGIGVLLDTNYNLFYWYEGLDHALQCVDYIKADGQNIGCRFPYLEASDYKDFYICVNGSSENKPIRSSYFTFQLQNIVKPLPPVYLTFTRESSCEIKLKWSIPLGPIPARCFDYEIEIREDDTTLVTATVENETYTLKTTNETRQLCFVVRSKVNIYCSDDGIWSEWSDKQCWEGEDLSKKTLLRFWLPFGFILILVIFVTGLLLRKPNTYPKMIPEFFCDT

Accordingly, a preferred complex according to the present inventioncomprises an amino acid sequence according to SEQ ID NO: 61 or afragment or a variant thereof as described herein.

Several epitopes of IL13Ralpha2are known to the skilled person. Apreferred IL13Ralpha2 epitope, which is preferably comprised by thecomplex according to the present invention, includes the followingepitope (the epitope sequence shown in the following is a fragment ofthe above IL13Ralpha2 sequence and is, thus, shown in the aboveIL13Ralpha2 sequence underlined; the following epitope sequence mayrefer to one epitope or more than one (overlapping) epitopes):

[SEQ ID NO: 62] LPFGFIL

Accordingly, a preferred complex according to the present inventioncomprises an amino acid sequence according to SEQ ID NO: 62.

CMV

Human cytomegalovirus (CMV), a member of the herpes virus family,persists after a usually unrecognized infection lifelong in the body.CMV is the most frequent opportunistic CNS infection in severelyimmunocompromised patients. Postmortem CMV nucleic acid can be detectedin the CNS of more than 20% of individuals without CNS diseases,confirming tropism of CMV to CNS cells and indicating that CMV maypersist in the CNS lifelong without causing (obvious) harm. However,expression of CMV proteins and oligonucleotides was also identified in ahigh percentage of gliomas (Cobbs C S, Harkins L, Samanta M, et al.Human cytomegalovirus infection and expression in human malignantglioma. Cancer Res. 2002; 62:3347-3350).

Preferred CMV antigens are those as described in WO 2009/155535, such asa polypeptide, or an immunogenic fragment thereof, selected from thegroup consisting of capsid polypeptides, tegument polypeptides, andenvelope polypeptides; preferably selected from the group consisting ofphosphoprotein unique long 83 (ppUL83; a/k/a pp65), glycoprotein UL55(gpUL55; a/k/a gB), UL123 immediate early 1 (IEI) protein, UL122 IE2protein, ULI I IA (a/k/a mtrII), US28, ppUL32, ppUL65, ppUL80a, ppUL82,ppUL98a, ppUL99, gpUL4 (a/k/a gp48), gpULIβ, gpUL18 (a/k/a MHC), gpUL75(a/k/a gH), gpULIOO, gpULHO (a/k/a gM), gpUL115 (a/k/a gL), pUL46,pUL48, pUL56, pUL86 (a/k/a MCP), glycoprotein unique short 10 (gpUSIO),gpUSI 1, glycoprotein complex II (gcII), gp65, and gp93. Preferably, theCMV antigen is a polypeptide, or an immunogenic fragment thereof, whichis encoded by a gene corresponding to the CMV strain shown by GENBANKAccession No. BK000394.2 or XI 7403.1. Moreover, WO 2009/155535 alsodescribes preferred CMV epitopes such as the peptides comprising theamino acid sequences as described by Trivedi et al., Blood, 105:2793(2005) and U.S. Patent Application Publication No. 2005/0019344 andother CMV epitopes as described in WO 2009/155535.

EGFRvIII

The epidermal growth factor receptor (EGFR; ErbB-1; HER1 in humans) isthe cell-surface receptor for members of the epidermal growth factorfamily (EGF-family) of extracellular protein ligands. EGFRvIII is amutated form of EGFR, which is known to play a prominent role intumorigenesis. Namely, EGFRvIII is a constitutively activated tyrosinekinase derived from a deletion mutation in EGFR. EGFRvIII is largelyoverexpressed by glioblastoma cells (Saikali, S., et al., Expression ofnine tumour antigens in a series of human glioblastoma multiforme:interest of EGFRvIII, IL-13Ralpha2, gp100 and TRP-2 for immunotherapy. JNeurooncol, 2007. 81(2): p. 139-48), which acquire enhanced capacity forunregulated growth, survival, invasion, and recruitment of new tumorblood vessels.

Several epitopes of EGFRvIII are known to the skilled person. Apreferred EGFRvIII epitope, which is preferably comprised by the complexaccording to the present invention, includes the following epitope (thefollowing epitope sequence may refer to one epitope or more than one(overlapping) epitopes):

[SEQ ID NO: 71] LEEKKGNYVVTDHC

Accordingly, a preferred complex according to the present inventioncomprises an amino acid sequence according to SEQ ID NO: 71.

EphA2

Ephrins and their receptors (ephrin receptors, “Ephs”) belong to asubfamily of proteins involved in crucial processes occurring duringembryonic development of the central nervous system, including axonmapping, cell migration, and angiogenesis. Recent findings suggest thatEph/ephrin are overexpressed by glioma cells and their signaling affectsglioma cell growth, migration, and invasion (Nakada, M., Y. Hayashi, andJ. Hamada, Role of Eph/ephrin tyrosine kinase in malignant glioma. NeuroOncol, 2011. 13(11): p. 1163-70). 16 ephrin receptors (Ephs) have beenidentified. The ephrin receptors are divided into two groups based onthe similarity of their extracellular domain sequences and theiraffinities for binding ephrin-A and ephrin-B ligands. EPH receptor A2(ephrin type-A receptor 2) binds ephrin-A ligands. The amino acidsequence of EphA2 is shown in the following:

[SEQ ID NO: 72] MELQAARACFALLWGCALAAAAAAQGKEVVLLDFAAAGGELGWLTHPYGKGWDLMQNIMNDMPIYMYSVCNVMSGDQDNWLRTNWVYRGEAERIFIELKFTVRDCNSFPGGASSCKETFNLYYAESDLDYGTNFQKRLFTKIDTIAPDEITVSSDFEARHVKLNVEERSVGPLTRKGFYLAFQDIGACVALLSVRVYYKKCPELLQGLAHFPETIAGSDAPSLATVAGTCVDHAVVPPGGEEPRMHCAVDGEWLVPIGQCLCQAGYEKVEDACQACSPGFFKFEASESPCLECPEHTLPSPEGATSCECEEGFFRAPQDPASMPCTRPPSAPHYLTAVGMGAKVELRWTPPQDSGGREDIVYSVTCEQCWPESGECGPCEASVRYSEPPHGLTRTSVTVSDLEPHMNYTFTVEARNGVSGLVTSRSFRTASVSINQTEPPKVRLEGRSTTSLSVSWSIPPPQQSRVWKYEVTYRKKGDSNSYNVRRTEGFSVTLDDLAPDTTYLVQVQALTQEGQGAGSKVHEFQTLSPEGSGNLAVIGGVAVGVVLLLVLAGVGFFIHRRRKNQRARQSPEDVYFSKSEQLKPLKTYVDPHTYEDPNQAVLKFTTEIHPSCVTRQKVIGAGEFGEVYKGMLKTSSGKKEVPVAIKTLKAGYTEKQRVDFLGEAGIMGQFSHHNIIRLEGVISKYKPMMIITEYMENGALDKFLREKDGEFSVLQLVGMLRGIAAGMKYLANMNYVHRDLAARNILVNSNLVCKVSDFGLSRVLEDDPEATYTTSGGKIPIRWTAPEAISYRKFTSASDVWSFGIVMWEVMTYGERPYWELSNHEVMKAINDGFRLPTPMDCPSAIYQLMMQCWQQERARRPKFADIVSILDKLIRAPDSLKTLADFDPRVSIRLPSTSGSEGVPFRTVSEWLESIKMQQYTEHFMAAGYTAIEKVVQMTNDDIKRIGVRLPGHQKRIAYSLLGLKDQVNTVGIPI

Accordingly, a preferred complex according to the present inventioncomprises an amino acid sequence according to SEQ ID NO: 72 or afragment or a variant thereof as described herein. As described above,suitable cancer/tumor epitopes of EphA2 are known from the literature orcan be identified by using cancer/tumor epitope databases, e.g. from vander Bruggen P, Stroobant V, Vigneron N, Van den Eynde B. Peptidedatabase: T cell-defined tumor antigens. Cancer Immun 2013; URL:http://www.cancerimmunity.org/peptide/, wherein human tumor antigensrecognized by CD4+ or CD8+ T cells are classified into four major groupson the basis of their expression pattern, or from the database“Tantigen” (TANTIGEN version 1.0, Dec. 1, 2009; developed byBioinformatics Core at Cancer Vaccine Center, Dana-Farber CancerInstitute; URL: http://cvc.dfci.harvard.edu/tadb/).

Gp100

Glycoprotein 100, gp100 or Melanocyte protein PMEL is 661 amino acidslong and is a type I transmembrane glycoprotein enriched in melanosomes,which are the melanin-producing organelles in melanocytes. Gp100 isinvolved in melanosome maturation. The gp100 protein is a melanomaantigen i.e. a tumor-associated antigen. The amino acid sequence ofgp100 is shown in the following:

[SEQ ID NO: 73] MDLVLKRCLLHLAVIGALLAVGATKVPRNQDWLGVSRQLRTKAWNRQLYPEWTEAQRLDCWRGGQVSLKVSNDGPTLIGANASFSIALNFPGSQKVLPDGQVIWVNNTIINGSQVWGGQPVYPQETDDACIFPDGGPCPSGSWSQKRSFVYVWKTWGQYWQVLGGPVSGLSIGTGRAMLGTHTMEVTVYHRRGSRSYVPLAHSSSAFTITDQVPFSVSVSQLRALDGGNKHFLRNQPLTFALQLHDPSGYLAEADLSYTWDFGDSSGTLISRALVVTHTYLEPGPVTAQVVLQAAIPLTSCGSSPVPGTTDGHRPTAEAPNTTAGQVPTTEVVGTTPGQAPTAEPSGTTSVQVPTTEVISTAPVQMPTAESTGMTPEKVPVSEVMGTTLAEMSTPEATGMTPAEVSIVVLSGTTAAQVTTTEWVETTARELPIPEPEGPDASSIMSTESITGSLGPLLDGTATLRLVKRQVPLDCVLYRYGSFSVTLDIVQGIESAEILQAVPSGEGDAFELTVSCQGGLPKEACMEISSPGCQPPAQRLCQPVLPSPACQLVLHQILKGGSGTYCLNVSLADTNSLAVVSTQLIMPGQEAGLGQVPLIVGILLVLMAVVLASLIYRRRLMKQDFSVPQLPHSSSHWLRLPRIFCSCPIG ENSPLLSGQQV

Accordingly, a preferred complex according to the present inventioncomprises an amino acid sequence according to SEQ ID NO: 73 or afragment or a variant thereof as described herein. As described above,suitable cancer/tumor epitopes of gp100 are known from the literature orcan be identified by using cancer/tumor epitope databases, e.g. from vander Bruggen P, Stroobant V, Vigneron N, Van den Eynde B. Peptidedatabase: T cell-defined tumor antigens. Cancer lmmun 2013; URL:http://www.cancerimmunity.org/peptide/, wherein human tumor antigensrecognized by CD4+ or CD8+ T cells are classified into four major groupson the basis of their expression pattern, or from the database“Tantigen” (TANTIGEN version 1.0, Dec 1, 2009; developed byBioinformatics Core at Cancer Vaccine Center, Dana-Farber CancerInstitute; URL: http://cvc.dfci.harvard.edu/tadb/).

hTert

Telomerase reverse transcriptase (abbreviated to TERT, or hTERT inhumans) is a catalytic subunit of the enzyme telomerase, which, togetherwith the telomerase RNA component (TERC), comprises the most importantunit of the telomerase complex. Telomerases are part of a distinctsubgroup of RNA-dependent polymerases. Telomerase lengthens telomeres inDNA strands, thereby allowing senescent cells that would otherwisebecome postmitotic and undergo apoptosis to exceed the Hayflick limitand become potentially immortal, as is often the case with cancerouscells. The amino acid sequence of hTert is shown in the following:

[SEQ ID NO: 74] MPRAPRCRAVRSLLRSHYREVLPLATFVRRLGPQGWRLVQRGDPAAFRALVAQCLVCVPWDARPPPAAPSFRQVSCLKELVARVLQRLCERGAKNVLAFGFALLDGARGGPPEAFTTSVRSYLPNTVTDALRGSGAWGLLLRRVGDDVLVHLLARCALFVLVAPSCAYQVCGPPLYQLGAATQARPPPHASGPRRRLGCERAWNHSVREAGVPLGLPAPGARRRGGSASRSLPLPKRPRRGAAPEPERTPVGQGSWAHPGRTRGPSDRGFCVVSPARPAEEATSLEGALSGTRHSHPSVGRQHHAGPPSTSRPPRPWDTPCPPVYAETKHFLYSSGDKEQLRPSFLLSSLRPSLTGARRLVETIFLGSRPWMPGTPRRLPRLPQRYWQMRPLFLELLGNHAQCPYGVLLKTHCPLRAAVTPAAGVCAREKPQGSVAAPEEEDTDPRRLVQLLRQHSSPWQVYGFVRACLRRLVPPGLWGSRHNERRFLRNTKKFISLGKHAKLSLQELTWKMSVRDCAWLRRSPGVGCVPAAEHRLREEILAKFLHWLMSVYVVELLRSFFYVTETTFQKNRLFFYRKSVWSKLQSIGIRQHLKRVQLRELSEAEVRQHREARPALLTSRLRFIPKPDGLRPIVNMDYVVGARTFRREKRAERLTSRVKALFSVLNYERARRPGLLGASVLGLDDIHRAWRTFVLRVRAQDPPPELYFVKVDVTGAYDTIPQDRLTEVIASIIKPQNTYCVRRYAVVQKAAHGHVRKAFKSHVSTLTDLQPYMRQFVAHLQETSPLRDAVVIEQSSSLNEASSGLFDVFLRFMCHHAVRIRGKSYVQCQGIPQGSILSTLLCSLCYGDMENKLFAGIRRDGLLLRLVDDFLLVTPHLTHAKTFLRTLVRGVPEYGCVVNLRKTVVNFPVEDEALGGTAFVQMPAHGLFPWCGLLLDTRTLEVQSDYSSYARTSIRASLTFNRGFKAGRNMRRKLFGVLRLKCHSLFLDLQVNSLQTVCTNIYKILLLQAYRFHACVLQLPFHQQVWKNPTFFLRVISDTASLCYSILKAKNAGMSLGAKGAAGPLPSEAVQWLCHQAFLLKLTRHRVTYVPLLGSLRTAQTQLSRKLPGTTLTALEAAANPALPSDFKTILD

Accordingly, a preferred complex according to the present inventioncomprises an amino acid sequence according to SEQ ID NO: 74 or afragment or a variant thereof as described herein. As described above,suitable cancer/tumor epitopes of hTert are known from the literature orcan be identified by using cancer/tumor epitope databases, e.g. from vander Bruggen P, Stroobant V, Vigneron N, Van den Eynde B. Peptidedatabase: T cell-defined tumor antigens. Cancer lmmun 2013; URL:http://www.cancerimmunity.org/peptide/, wherein human tumor antigensrecognized by CD4+ or CD8+ T cells are classified into four major groupson the basis of their expression pattern, or from the database“Tantigen” (TANTIGEN version 1.0, Dec. 1, 2009; developed byBioinformatics Core at Cancer Vaccine Center, Dana-Farber CancerInstitute; URL: http://cvc.dfci.harvard.edu/tadb/).

TRP-2

Tyrosinase related protein 2 (TRP-2, also known as “L-DOPAchrometautomerase”) is a melanogenic enzyme. TRP-2 regulates a switch thatcontrols the proportion of carboxylated subunits in the melaninbiopolymer. Thus, TRP-2 is a melanosome protein involved in melaninproduction. It is also overexpressed in glioblastoma cells. The aminoacid sequence of TRP-2 is shown in the following:

[SEQ ID NO: 75] MSPLWWGFLLSCLGCKILPGAQGQFPRVCMTVDSLVNKECCPRLGAESANVCGSQQGRGQCTEVRADTRPWSGPYILRNQDDRELWPRKFFHRTCKCTGNFAGYNCGDCKFGWTGPNCERKKPPVIRQNIHSLSPQEREQFLGALDLAKKRVHPDYVITTQHWVGLLGPNGTQPQFANCSVYDFFVWLHYYSVRDTLLGGFFPWLKVYYYRFVIGLRVWQWEVISCKLIKRATTRQP

Accordingly, a preferred complex according to the present inventioncomprises an amino acid sequence according to SEQ ID NO: 75 or afragment or a variant thereof as described herein. As described above,suitable cancer/tumor epitopes of TRP-2 are known from the literature orcan be identified by using cancer/tumor epitope databases, e.g. from vander Bruggen P, Stroobant V, Vigneron N, Van den Eynde B. Peptidedatabase: T cell-defined tumor antigens. Cancer Immun 2013; URL:http://www.cancerimmunity.org/peptide/, wherein human tumor antigensrecognized by CD4+ or CD8+ T cells are classified into four major groupson the basis of their expression pattern, or from the database“Tantigen” (TANTIGEN version 1.0, Dec. 1, 2009; developed byBioinformatics Core at Cancer Vaccine Center, Dana-Farber CancerInstitute; URL: http://cvc.dfci.harvard.edu/tadb/).

YKL-40

YKL-40 (also known as human cartilage glycoprotein-39 (HC gp-39) and asChitinase-3-like protein 1 (CHI3L1)), is a secreted glycoprotein and amember of family 18 glycosyl hydrolases. The name YKL-40 is derived fromthe three N-terminal amino acids present on the secreted form and itsmolecular mass. YKL-40 is secreted by various cell types, includingchondrocytes, synovial cells, macrophages and some types of cancercells. The amino acid sequence of YKL-40 is shown in the following:

[SEQ ID NO: 76] MGVKASQTGFVVLVLLQCCSAYKLVCYYTSWSQYREGDGSCFPDALDRFLCTHIIYSFANISNDHIDTWEWNDVTLYGMLNTLKNRNPNLKTLLSVGGWNFGSQRFSKIASNTQSRRTFIKSVPPFLRTHGFDGLDLAWLYPGRRDKQHFTTLIKEMKAEFIKEAQPGKKQLLLSAALSAGKVTIDSSYDIAKISQHLDFISIMTYDFHGAWRGTTGHHSPLFRGQEDASPDRFSNTDYAVGYMLRLGAPASKLVMGIPTFGRSFTLASSETGVGAPISGPGIPGRFTKEAGTLAYYEICDFLRGATVHRILGQQVPYATKGNQWVGYDDQESVKSKVQYLKDRQLAGAM

Accordingly, a preferred complex according to the present inventioncomprises an amino acid sequence according to SEQ ID NO: 76 or afragment or a variant thereof as described herein. As described above,suitable cancer/tumor epitopes of YKL-40 are known from the literatureor can be identified by using cancer/tumor epitope databases, e.g. fromvan der Bruggen P, Stroobant V, Vigneron N, Van den Eynde B. Peptidedatabase: T cell-defined tumor antigens. Cancer lmmun 2013; URL:http://www.cancerimmunity.org/peptide/, wherein human tumor antigensrecognized by CD4+ or CD8+ T cells are classified into four major groupson the basis of their expression pattern, or from the database“Tantigen” (TANTIGEN version 1.0, Dec. 1, 2009; developed byBioinformatics Core at Cancer Vaccine Center, Dana-Farber CancerInstitute; URL: http://cvc.dfci.harvard.edu/tadb/).

Brevican

Brevican core protein is a protein that in humans is encoded by theBCANgene. Brevican is a member of the lectican protein family. Brevicanis localised to the surface of neurons in the brain. In melanocyticcells, BCAN gene expression may be regulated by MITF. The amino acidsequence of brevican is shown in the following:

[SEQ ID NO: 77] MAQLFLPLLAALVLAQAPAALADVLEGDSSEDRAFRVRIAGDAPLQGVLGGALTIPCHVHYLRPPPSRRAVLGSPRVKWTFLSRGREAEVLVARGVRVKVNEAYRFRVALPAYPASLTDVSLALSELRPNDSGIYRCEVQHGIDDSSDAVEVKVKGVVFLYREGSARYAFSFSGAQEACARIGAHIATPEQLYAAYLGGYEQCDAGWLSDQTVRYPIQTPREACYGDMDGFPGVRNYGVVDPDDLYDVYCYAEDLNGELFLGDPPEKLTLEEARAYCQERGAEIATTGQLYAAWDGGLDHCSPGWLADGSVRYPIVTPSQRCGGGLPGVKTLFLFPNQTGFPNKHSRFNVYCFRDSAQPSAIPEASNPASNPASDGLEAIVTVTETLEELQLPQEATESESRGAIYSIPIMEDGGGGSSTPEDPAEAPRTLLEFETQSMVPPTGFSEEEGKALEEEEKYEDEEEKEEEEEEEEVEDEALWAWPSELSSPGPEASLPTEPAAQEESLSQAPARAVLQPGASPLPDGESEASRPPRVHGPPTETLPTPRERNLASPSPSTLVEAREVGEATGGPELSGVPRGESEETGSSEGAPSLLPATRAPEGTRELEAPSEDNSGRTAPAGTSVQAQPVLPTDSASRGGVAVVPASGDCVPSPCHNGGTCLEEEEGVRCLCLPGYGGDLCDVGLRFCNPGWDAFQGACYKHFSTRRSWEEAETQCRMYGAHLASISTPEEQDFINNRYREYQWIGLNDRTIEGDFLWSDGVPLLYENWNPGQPDSYFLSGENCVVMVWHDQGQWSDVPCNYHLSYTCKMGLVSCGPPPELPLAQVFGRPRLRYEVDTVLRYRCREGLAQRNLPLIRCQENGRWEAPQISCVPRRPARALHPEEDPEGRQGRLLGRWKAL LIPPSSPMPGP

Accordingly, a preferred complex according to the present inventioncomprises an amino acid sequence according to SEQ ID NO: 77 or afragment or a variant thereof as described herein. As described above,suitable cancer/tumor epitopes of brevican are known from the literatureor can be identified by using cancer/tumor epitope databases, e.g. fromvan der Bruggen P, Stroobant V, Vigneron N, Van den Eynde B. Peptidedatabase: T cell-defined tumor antigens. Cancer Immun 2013; URL:http://www.cancerimmunity.org/peptide/, wherein human tumor antigensrecognized by CD4+ or CD8+ T cells are classified into four major groupson the basis of their expression pattern, or from the database“Tantigen” (TANTIGEN version 1.0, Dec. 1, 2009; developed byBioinformatics Core at Cancer Vaccine Center, Dana-Farber CancerInstitute; URL: http://cvc.dfci.harvard.edu/tadb/).

Neuroligin 4

Neuroligin (NLGN), a type I membrane protein, is a cell adhesion proteinon the postsynaptic membrane that mediates the formation and maintenanceof synapses between neurons. Neuroligins act as ligands for β-Neurexins,which are cell adhesion proteins located presynaptically. Neuroliginsalso affect the properties of neural networks by specifying synapticfunctions, and they mediate signalling by recruiting and stabilizing keysynaptic components. Neuroligins interact with other postsynapticproteins to localize neurotransmitter receptors and channels in thepostsynaptic density as the cell matures. The amino acid sequence ofneuroligin 4 is shown in the following:

[SEQ ID NO: 78] MSRPQGLLWLPLLFTPVCVMLNSNVLLWLTALAIKFTLIDSQAQYPVVNTNYGKIRGLRTPLPNEILGPVEQYLGVPYASPPTGERRFQPPEPPSSWTGIRNTTQFAAVCPQHLDERSLLHDMLPIWFTANLDTLMTYVQDQNEDCLYLNIYVPTEDDIHDQNSKKPVMVYIHGGSYMEGTGNMIDGSILASYGNVIVITINYRLGILGFLSTGDQAAKGNYGLLDQIQALRWIEENVGAFGGDPKRVTIFGSGAGASCVSLLTLSHYSEGLFQKAIIQSGTALSSWAVNYQPAKYTRILADKVGCNMLDTTDMVECLRNKNYKELIQQTITPATYHIAFGPVIDGDVIPDDPQILMEQGEFLNYDIMLGVNQGEGLKFVDGIVDNEDGVTPNDFDFSVSNFVDNLYGYPEGKDTLRETIKFMYTDWADKENPETRRKTLVALFTDHQWVAPAVATADLHAQYGSPTYFYAFYHHCQSEMKPSWADSAHGDEVPYVFGIPMIGPTELFSCNFSKNDVMLSAVVMTYWTNFAKTGDPNQPVPQDTKFIHTKPNRFEEVAWSKYNPKDQLYLHIGLKPRVRDHYRATKVAFWLELVPHLHNLNEIFQYVSTTTKVPPPDMTSFPYGTRRSPAKIWPTTKRPAITPANNPKHSKDPHKTGPEDTTVLIETKRDYSTELSVTIAVGASLLFLNILAFAALYYKKDKRRHETHRRPSPQRNTTNDIAHIQNEEIMSLQMKQLEHDHECESLQAHDTLRLTCPPDYTLTLRRSPDDIPLMTPNTITMIPNTLTGMQPLHTFNTFSG GQNSTNLPHGHSTTRV

Accordingly, a preferred complex according to the present inventioncomprises an amino acid sequence according to SEQ ID NO: 78 or afragment or a variant thereof as described herein. As described above,suitable cancer/tumor epitopes of neuroligin 4 are known from theliterature or can be identified by using cancer/tumor epitope databases,e.g. from van der Bruggen P, Stroobant V, Vigneron N, Van den Eynde B.Peptide database: T cell-defined tumor antigens. Cancer Immun 2013; URL:http://www.cancerimmunity.org/peptide/, wherein human tumor antigensrecognized by CD4+ or CD8+ T cells are classified into four major groupson the basis of their expression pattern, or from the database“Tantigen” (TANTIGEN version 1.0, Dec 1, 2009; developed byBioinformatics Core at Cancer Vaccine Center, Dana-Farber CancerInstitute; URL: http://cvc.dfci.harvard.edu/tadb/).

PTPRz1

Receptor-type tyrosine-protein phosphatase zeta also known as phosphacanis a single-pass type I membrane protein with two cytoplasmictyrosine-protein phosphatase domains, an alpha-carbonic anhydrase domainand a fibronectin type III domain and belongs to the receptor tyrosinephosphatase family. Both, the protein and transcript are overexpressedin glioblastoma cells, promoting their haptotactic migration. The aminoacid sequence of PTPRz1 is shown in the following:

[SEQ ID NO: 79] MRILKRFLACIQLLCVCRLDWANGYYRQQRKLVEEIGWSYTGALNQKNWGKKYPTCNSPKQSPINIDEDLTQVNVNLKKLKFQGWDKTSLENTFIHNTGKTVEINLTNDYRVSGGVSEMVFKASKITFHWGKCNMSSDGSEHSLEGQKFPLEMQIYCFDADRFSSFEEAVKGKGKLRALSILFEVGTEENLDFKAIIDGVESVSRFGKQAALDPFILLNLLPNSTDKYYIYNGSLTSPPCTDTVDWIVFKDTVSISESQLAVFCEVLTMQQSGYVMLMDYLQNNFREQQYKFSRQVFSSYTGKEEIHEAVCSSEPENVQADPENYTSLLVTWERPRVVYDTMIEKFAVLYQQLDGEDQTKHEFLTDGYQDLGAILNNLLPNMSYVLQIVAICTNGLYGKYSDQLIVDMPTDNPELDLFPELIGTEEIIKEEEEGKDIEEGAIVNPGRDSATNQIRKKEPQISTTTHYNRIGTKYNEAKTNRSPTRGSEFSGKGDVPNTSLNSTSQPVTKLATEKDISLTSQTVTELPPHTVEGTSASLNDGSKTVLRSPHMNLSGTAESLNTVSITEYEEESLLTSFKLDTGAEDSSGSSPATSAIPFISENISQGYIFSSENPETITYDVLIPESARNASEDSTSSGSEESLKDPSMEGNVWFPSSTDITAQPDVGSGRESFLQTNYTEIRVDESEKTTKSFSAGPVMSQGPSVTDLEMPHYSTFAYFPTEVTPHAFTPSSRQQDLVSTVNVVYSQTTQPVYNGETPLQPSYSSEVFPLVTPLLLDNQILNTTPAASSSDSALHATPVFPSVDVSFESILSSYDGAPLLPFSSASFSSELFRHLHTVSQILPQVTSATESDKVPLHASLPVAGGDLLLEPSLAQYSDVLSTTHAASETLEFGSESGVLYKTLMFSQVEPPSSDAMMHARSSGPEPSYALSDNEGSQHIFTVSYSSAIPVHDSVGVTYQGSLFSGPSHIPIPKSSLITPTASLLQPTHALSGDGEWSGASSDSEFLLPDTDGLTALNISSPVSVAEFTYTTSVFGDDNKALSKSEIIYGNETELQIPSFNEMVYPSESTVMPNMYDNVNKLNASLQETSVSISSTKGMFPGSLAHTTTKVFDHEISQVPENNFSVQPTHTVSQASGDTSLKPVLSANSEPASSDPASSEMLSPSTQLLFYETSASFSTEVLLQPSFQASDVDTLLKTVLPAVPSDPILVETPKVDKISSTMLHLIVSNSASSENMLHSTSVPVFDVSPTSHMHSASLQGLTISYASEKYEPVLLKSESSHQVVPSLYSNDELFQTANLEINQAHPPKGRHVFATPVLSIDEPLNTLINKLIHSDEILTSTKSSVTGKVFAGIPTVASDTFVSTDHSVPIGNGHVAITAVSPHRDGSVTSTKLLFPSKATSELSHSAKSDAGLVGGGEDGDTDDDGDDDDDDRGSDGLSIHKCMSCSSYRESQEKVMNDSDTHENSLMDQNNPISYSLSENSEEDNRVTSVSSDSQTGMDRSPGKSPSANGLSQKHNDGKEENDIQTGSALLPLSPESKAWAVLTSDEESGSGQGTSDSLNENETSTDFSFADTNEKDADGILAAGDSEITPGFPQSPTSSVTSENSEVFHVSEAEASNSSHESRIGLAEGLESEKKAVIPLVIVSALTFICLVVLVGILIYWRKCFQTAHFYLEDSTSPRVISTPPTPIFPISDDVGAIPIKHFPKHVADLHASSGFTEEFETLKEFYQEVQSCTVDLGITADSSNHPDNKHKNRYINIVAYDHSRVKLAQLAEKDGKLTDYINANYVDGYNRPKAYIAAQGPLKSTAEDFWRMIWEHNVEVIVMITNLVEKGRRKCDQYWPADGSEEYGNFLVTQKSVQVLAYYTVRNFTLRNTKIKKGSQKGRPSGRVVTQYHYTQWPDMGVPEYSLPVLTFVRKAAYAKRHAVGPVVVHCSAGVGRTGTYIVLDSMLQQIQHEGTVNIFGFLKHIRSQRNYLVQTEEQYVFIHDTLVEAILSKETEVLDSHIHAYVNALLIPGPAGKTKLEKQFQLLSQSNIQQSDYSAALKQCNREKNRTSSIIPVERSRVGISSLSGEGTDYINASYMGYYQSNEFIITQHPLLHTIKDFWRMIWDHNAQLVVMIPDGQNMAEDEFVYWPNKDEPINCESFKVTLMAEEHKCLSNEEKLIIQDFILEATQDDYVLEVRHFQCPKWPNPDSPISKTFELISVIKEEAANRDGPMIVHDEHGGVTAGTFCALTTLMHQLEKENSVDVYQVAKMINLMRPGVFADIEQYQFLYKVILSLVSTRQEENPSTSLDSNGAA LPDGNIAESLESLV

Accordingly, a preferred complex according to the present inventioncomprises an amino acid sequence according to SEQ ID NO: 79 or afragment or a variant thereof as described herein. As described above,suitable cancer/tumor epitopes of PTPRz1 are known from the literatureor can be identified by using cancer/tumor epitope databases, e.g. fromvan der Bruggen P, Stroobant V, Vigneron N, Van den Eynde B. Peptidedatabase: T cell-defined tumor antigens. Cancer Immun 2013; URL:http://www.cancerimmunity.org/peptide/, wherein human tumor antigensrecognized by CD4+ or CD8+ T cells are classified into four major groupson the basis of their expression pattern, or from the database“Tantigen” (TANTIGEN version 1.0, Dec. 1, 2009; developed byBioinformatics Core at Cancer Vaccine Center, Dana-Farber CancerInstitute; URL: http://cvc.dfci.harvard.edu/tadb/).

Preferably, the complex according to the present invention comprises atleast one tumor epitope, which is an epitope of an antigen selected fromthe group consisting of EpCAM, MUC-1, survivin, CEA, KRas, MAGE-A3,IL13Ralpha2, EGFRvIII, EphA2, Her2/neu, TRP-2, brevican, neuroligin 4and PTPRz1, such as an epitope according to any of SEQ ID NOs 48, 50,51, 53, 55, 56, 58, 60, 62 and 71; more preferably the at least onetumor epitope is an epitope of an antigen selected from the groupconsisting of EpCAM, MUC-1, survivin, CEA, KRas, MAGE-A3, EGFRvIII,EphA2, IL-13Rα2, TRP-2 and brevican such as an epitope according to anyof SEQ ID NOs 48, 50, 51, 53, 55, 56, 58, 60, 62 and 71; and even morepreferably the at least one tumor epitope is an epitope of an antigenselected from the group consisting of EpCAM, MUC-1, surviving, CEA,EGFRvIII, EphA2 and brevican, such as an epitope according to any of SEQID NOs 48, 50, 51, 53, 55, 56 and 71.

It is also preferred that the complex according to the present inventioncomprises

-   -   one or more epitopes of EpCAM (such as the epitope according to        SEQ ID NO: 48) or functional sequence variants thereof;    -   one or more epitopes of MUC-1 (such as the epitope according to        SEQ ID NO: 50 and/or the epitope according to SEQ ID NO: 51) or        functional sequence variants thereof;    -   one or more epitopes of survivin (such as the epitope according        to SEQ ID NO: 53) or functional sequence variants thereof;    -   one or more epitopes of CEA (such as the epitope according to        SEQ ID NO: 55 and/or the epitope according to SEQ ID NO: 56) or        functional sequence variants thereof;    -   one or more epitopes of KRas (such as the epitope according to        SEQ ID NO: 58) or functional sequence variants thereof;    -   one or more epitopes of MAGE-A3 (such as the epitope according        to SEQ ID NO: 60) or functional sequence variants thereof;    -   one or more epitopes of EGFRvIII (such as the epitope according        to SEQ ID NO: 71) or functional sequence variants thereof;    -   one or more epitopes of EphA2 or functional sequence variants        thereof;    -   one or more epitopes of Her2/neu or functional sequence variants        thereof;    -   one or more epitopes of IL-13Rα2 (such as the epitope according        to SEQ ID NO: 62) or functional sequence variants thereof;    -   one or more epitopes of TRP-2 or functional sequence variants        thereof;    -   one or more epitopes of brevican or functional sequence variants        thereof;    -   one or more epitopes of neuroligin 4 or functional sequence        variants thereof; and/or    -   one or more epitopes of PTPRz1 or functional sequence variants        thereof.

As described above, further epitopes of those antigens (in addition tothe exemplified epitopes) can easily be retrieved from cancer/tumorepitope databases, e.g. from van der Bruggen P, Stroobant V, Vigneron N,Van den Eynde B. Peptide database: T cell-defined tumor antigens. CancerImmun 2013; URL: http://www.cancerimmunity.org/peptide/, or from thedatabase “Tantigen” (TANTIGEN version 1.0, Dec. 1, 2009; developed byBioinformatics Core at Cancer Vaccine Center, Dana-Farber CancerInstitute; URL: http://cvc.dfci.harvard.edu/tadb/).

A “sequence variant” is as defined above, namely a sequence variant hasan (amino acid) sequence which is at least 70%, at least 75%, preferablyat least 80%, more preferably at least 85%, even more preferably atleast 90%, particularly preferably at least 95%, most preferably atleast 99% identical to the reference sequence. A “functional” sequencevariant means in the context of an epitope, that the function as anepitope is not impaired or abolished. Preferably, however, the aminoacid sequence of an epitope of a cancer/tumor antigen as describedherein is not mutated and, thus, identical to the reference epitopesequence.

It is also preferred that the complex according to the present inventioncomprises

-   -   a fragment of EpCAM comprising one or more epitopes or a        functional sequence variant thereof;    -   a fragment of MUC-1 comprising one or more epitopes or a        functional sequence variant thereof;    -   a fragment of CEA comprising one or more epitopes or a        functional sequence variant thereof;    -   a fragment of KRas comprising one or more epitopes or a        functional sequence variant thereof; and/or    -   a fragment of MAGE-A3 comprising one or more epitopes or a        functional sequence variant thereof    -   a fragment of EGFRvIlI comprising one or more epitopes or a        functional sequence variant thereof;    -   a fragment of EphA2 (such as a fragment of the polypeptide        according to SEQ ID NO: 72) comprising one or more epitopes or a        functional sequence variant thereof;    -   a fragment of Her2/neu (such as a fragment of the polypeptide        according to SEQ ID NO: 70) comprising one or more epitopes or a        functional sequence variant thereof;    -   a fragment of IL-13Rα2 (such as a fragment of the polypeptide        according to SEQ ID NO: 61) comprising one or more epitopes or a        functional sequence variant thereof;    -   a fragment of survivin (such as a fragment of the polypeptide        according to SEQ ID NO: 52) comprising one or more epitopes or a        functional sequence variant thereof;    -   a fragment of TRP-2 (such as a fragment of the polypeptide        according to SEQ ID NO: 75) comprising one or more epitopes or a        functional sequence variant thereof;    -   a fragment of brevican (such as a fragment of the polypeptide        according to SEQ ID NO: 77) comprising one or more epitopes or a        functional sequence variant thereof;    -   a fragment of neuroligin 4 (such as a fragment of the        polypeptide according to SEQ ID NO: 78) comprising one or more        epitopes or a functional sequence variant thereof; and/or    -   a fragment of PTPRz1 (such as a fragment of the polypeptide        according to SEQ ID NO: 79) comprising one or more epitopes or a        functional sequence variant thereof.

As used herein, a “fragment” of an antigen comprises at least 10consecutive amino acids of the antigen, preferably at least 15consecutive amino acids of the antigen, more preferably at least 20consecutive amino acids of the antigen, even more preferably at least 25consecutive amino acids of the antigen and most preferably at least 30consecutive amino acids of the antigen. Accordingly, a fragment of EpCAMcomprises at least 10 consecutive amino acids of EpCAM (SEQ ID NO: 47),preferably at least 15 consecutive amino acids of EpCAM (SEQ ID NO: 47),more preferably at least 20 consecutive amino acids of EpCAM (SEQ ID NO:47), even more preferably at least 25 consecutive amino acids of EpCAM(SEQ ID NO: 47) and most preferably at least 30 consecutive amino acidsof EpCAM (SEQ ID NO: 47); a fragment of MUC-1 comprises at least 10consecutive amino acids of MUC-1 (SEQ ID NO: 49), preferably at least 15consecutive amino acids of MUC-1 (SEQ ID NO: 49), more preferably atleast 20 consecutive amino acids of MUC-1 (SEQ ID NO: 49), even morepreferably at least 25 consecutive amino acids of MUC-1 (SEQ ID NO: 49)and most preferably at least 30 consecutive amino acids of MUC-1 (SEQ IDNO: 49); a fragment of survivin comprises at least 10 consecutive aminoacids of survivin (SEQ ID NO: 52), preferably at least 15 consecutiveamino acids of survivin (SEQ ID NO: 52), more preferably at least 20consecutive amino acids of survivin (SEQ ID NO: 52), even morepreferably at least 25 consecutive amino acids of survivin (SEQ ID NO:52) and most preferably at least 30 consecutive amino acids of survivin(SEQ ID NO: 52); a fragment of CEA comprises at least 10 consecutiveamino acids of CEA (SEQ ID NO: 54), preferably at least 15 consecutiveamino acids of CEA (SEQ ID NO: 54), more preferably at least 20consecutive amino acids of CEA (SEQ ID NO: 54), even more preferably atleast 25 consecutive amino acids of CEA (SEQ ID NO: 54) and mostpreferably at least 30 consecutive amino acids of CEA (SEQ ID NO: 54); afragment of KRas comprises at least 10 consecutive amino acids of KRas(SEQ ID NO: 57), preferably at least 15 consecutive amino acids of KRas(SEQ ID NO: 57), more preferably at least 20 consecutive amino acids ofKRas (SEQ ID NO: 57), even more preferably at least 25 consecutive aminoacids of KRas (SEQ ID NO: 57) and most preferably at least 30consecutive amino acids of KRas (SEQ ID NO: 57); a fragment of MAGE-A3comprises at least 10 consecutive amino acids of MAGE-A3 (SEQ ID NO:59), preferably at least 15 consecutive amino acids of MAGE-A3 (SEQ IDNO: 59), more preferably at least 20 consecutive amino acids of MAGE-A3(SEQ ID NO: 59), even more preferably at least 25 consecutive aminoacids of MAGE-A3 (SEQ ID NO: 59) and most preferably at least 30consecutive amino acids of MAGE-A3 (SEQ ID NO: 59); a fragment ofEGFRvIII comprises at least 10 consecutive amino acids of EGFRvIII,preferably at least 15 consecutive amino acids of EGFRvIII, morepreferably at least 20 consecutive amino acids of EGFRvIII, even morepreferably at least 25 consecutive amino acids of EGFRvIII, and mostpreferably at least 30 consecutive amino acids of EGFRvIII; a fragmentof EphA2 comprises at least 10 consecutive amino acids of EphA2 (SEQ IDNO: 72), preferably at least 15 consecutive amino acids of EphA2 (SEQ IDNO: 72), more preferably at least 20 consecutive amino acids of EphA2(SEQ ID NO: 72), even more preferably at least 25 consecutive aminoacids of EphA2 (SEQ ID NO: 72) and most preferably at least 30consecutive amino acids of EphA2 (SEQ ID NO: 72); a fragment of Her2/neucomprises at least 10 consecutive amino acids of Her2/neu (SEQ ID NO:70), preferably at least 15 consecutive amino acids of Her2/neu (SEQ IDNO: 70), more preferably at least 20 consecutive amino acids of Her2/neu(SEQ ID NO: 70), even more preferably at least 25 consecutive aminoacids of Her2/neu (SEQ ID NO: 70) and most preferably at least 30consecutive amino acids of Her2/neu (SEQ ID NO: 70); a fragment ofIL-13Rα2 comprises at least 10 consecutive amino acids of IL-13Rα2 (SEQID NO: 61), preferably at least 15 consecutive amino acids of IL-13Rα2(SEQ ID NO: 61), more preferably at least 20 consecutive amino acids ofIL-13Rα2 (SEQ ID NO: 61), even more preferably at least 25 consecutiveamino acids of IL-13Rα2 (SEQ ID NO: 61) and most preferably at least 30consecutive amino acids of IL-13Rα2 (SEQ ID NO: 61); a fragment of TRP-2comprises at least 10 consecutive amino acids of TRP-2 (SEQ ID NO: 75),preferably at least 15 consecutive amino acids of TRP-2 (SEQ ID NO: 75),more preferably at least 20 consecutive amino acids of TRP-2 (SEQ ID NO:75), even more preferably at least 25 consecutive amino acids of TRP-2(SEQ ID NO: 75) and most preferably at least 30 consecutive amino acidsof TRP-2 (SEQ ID NO: 75); a fragment of brevican comprises at least 10consecutive amino acids of brevican (SEQ ID NO: 77), preferably at least15 consecutive amino acids of brevican (SEQ ID NO: 77), more preferablyat least 20 consecutive amino acids of brevican (SEQ ID NO: 77), evenmore preferably at least 25 consecutive amino acids of brevican (SEQ IDNO: 77) and most preferably at least 30 consecutive amino acids ofbrevican (SEQ ID NO: 77); a fragment of neuroligin 4 comprises at least10 consecutive amino acids of neuroligin 4 (SEQ ID NO: 78), preferablyat least 15 consecutive amino acids of neuroligin 4 (SEQ ID NO: 78),more preferably at least 20 consecutive amino acids of neuroligin 4 (SEQID NO: 78), even more preferably at least 25 consecutive amino acids ofneuroligin 4 (SEQ ID NO: 78) and most preferably at least 30 consecutiveamino acids of neuroligin 4 (SEQ ID NO: 78); and a fragment of PTPRz1comprises at least 10 consecutive amino acids of PTPRz1 (SEQ ID NO: 79),preferably at least 15 consecutive amino acids of PTPRz1 (SEQ ID NO:79), more preferably at least 20 consecutive amino acids of PTPRz1 (SEQID NO: 79), even more preferably at least 25 consecutive amino acids ofPTPRz1 (SEQ ID NO: 79) and most preferably at least 30 consecutive aminoacids of PTPRz1 (SEQ ID NO: 79).

A functional sequence variant of such a fragment has an (amino acid)sequence, which is at least 70%, at least 75%, preferably at least 80%,more preferably at least 85%, even more preferably at least 90%,particularly preferably at least 95%, most preferably at least 99%identical to the reference sequence, and the epitope function of atleast one, preferably all, epitope(s) comprised by the fragment ismaintained.

Preferably, such a complex comprises

-   -   one or more epitopes of EpCAM (such as the epitope according to        SEQ ID NO: 48) or functional sequence variants thereof;    -   one or more epitopes of CEA (such as the epitope according to        SEQ ID NO: 55 and/or the epitope according to SEQ ID NO: 56) or        functional sequence variants thereof; and    -   one or more epitopes of MAGE-A3 (such as the epitope according        to SEQ ID NO: 60) or functional sequence variants thereof.

Such a complex does preferably not comprise any epitope of HER-2, MUC-1,TOMM34, RNF 43, KOC1, VEGFR, βhCG, survivin, TGFβR2, p53, KRas, OGT,CASPS, COA-1, SART or IL13Ralpha2.

It is also preferred that such a complex comprises

-   -   one or more epitopes of EpCAM (such as the epitope according to        SEQ ID NO: 48) or functional sequence variants thereof;    -   one or more epitopes of MUC-1 (such as the epitope according to        SEQ ID NO: 50 and/or the epitope according to SEQ ID NO: 51) or        functional sequence variants thereof;    -   one or more epitopes of CEA (such as the epitope according to        SEQ ID NO: 55 and/or the epitope according to SEQ ID NO: 56) or        functional sequence variants thereof; and    -   one or more epitopes of MAGE-A3 (such as the epitope according        to SEQ ID NO: 60) or functional sequence variants thereof.

Such a complex does preferably not comprise any epitope of HER-2,TOMM34, RNF 43, KOC1, VEGFR, βhCG, survivin, TGFβR2, p53, KRas, OGT,CASP5, COA-1, SART or IL13Ralpha2.

It is also preferred that such a complex comprises

-   -   one or more epitopes of EpCAM (such as the epitope according to        SEQ ID NO: 48) or functional sequence variants thereof;    -   one or more epitopes of MUC-1 (such as the epitope according to        SEQ ID NO: 50 and/or the epitope according to SEQ ID NO: 51) or        functional sequence variants thereof;    -   one or more epitopes of CEA (such as the epitope according to        SEQ ID NO: 55 and/or the epitope according to SEQ ID NO: 56) or        functional sequence variants thereof; and    -   one or more epitopes of KRas (such as the epitope according to        SEQ ID NO: 58) or functional sequence variants thereof.

Such a complex does preferably not comprise any epitope of HER-2,TOMM34, RNF 43, KOC1, VEGFR, βhCG, survivin, TGFβR2, p53, OGT, CASP5,COA-1, MAGE, SART or IL13Ralpha2.

It is also preferred that such a complex comprises

-   -   one or more epitopes of EpCAM (such as the epitope according to        SEQ ID NO: 48) or functional sequence variants thereof;    -   one or more epitopes of survivin (such as the epitope according        to SEQ ID NO: 53) or functional sequence variants thereof;    -   one or more epitopes of CEA (such as the epitope according to        SEQ ID NO: 55 and/or the epitope according to SEQ ID NO: 56) or        functional sequence variants thereof; and    -   one or more epitopes of MAGE-A3 (such as the epitope according        to SEQ ID NO: 60) or functional sequence variants thereof.

Such a complex does preferably not comprise any epitope of HER-2, MUC-1,TOMM34, RNF 43, KOC1, VEGFR, βhCG, TGFβR2, p53, KRas, OGT, CASP5, COA-1,SART or IL13Ralpha2.

It is also preferred that such a complex comprises

-   -   one or more epitopes of MUC-1 (such as the epitope according to        SEQ ID NO: 50 and/or the epitope according to SEQ ID NO: 51) or        functional sequence variants thereof;    -   one or more epitopes of survivin (such as the epitope according        to SEQ ID NO: 53) or functional sequence variants thereof; and    -   one or more epitopes of MAGE-A3 (such as the epitope according        to SEQ ID NO: 60) or functional sequence variants thereof.

Such a complex does preferably not comprise any epitope of EpCAM, HER-2,TOMM34, RNF 43, KOC1, VEGFR, βhCG, CEA, TGFβR2, p53, KRas, OGT, CASP5,COA-1, SART or IL13Ralpha2.

More preferably, such a complex comprises

-   -   one or more epitopes of EpCAM (such as the epitope according to        SEQ ID NO: 48) or functional sequence variants thereof;    -   one or more epitopes of MUC-1 (such as the epitope according to        SEQ ID NO: 50 and/or the epitope according to SEQ ID NO: 51) or        functional sequence variants thereof;    -   one or more epitopes of survivin (such as the epitope according        to SEQ ID NO: 53) or functional sequence variants thereof;        and/or    -   one or more epitopes of CEA (such as the epitope according to        SEQ ID NO: 55 and/or the epitope according to SEQ ID NO: 56) or        functional sequence variants thereof.

Such a complex does preferably not comprise any epitope of HER-2,TOMM34, RNF 43, KOC1, VEGFR, βhCG, TGFβR2, p53, KRas, OGT, CASP5, COA-1,MAGE, SART or IL13Ralpha2.

Particularly preferably, such a complex comprises

-   -   one or more epitopes of EpCAM (such as the epitope according to        SEQ ID NO: 48) or functional sequence variants thereof;    -   one or more epitopes of MUC-1 (such as the epitope according to        SEQ ID NO: 50 and/or the epitope according to SEQ ID NO: 51) or        functional sequence variants thereof;    -   one or more epitopes of survivin (such as the epitope according        to SEQ ID NO: 53) or functional sequence variants thereof; and    -   one or more epitopes of CEA (such as the epitope according to        SEQ ID NO: 55 and/or the epitope according to SEQ ID NO: 56) or        functional sequence variants thereof.

Such a complex does preferably not comprise any epitope of HER-2,TOMM34, RNF 43, KOC1, VEGFR, βhCG, TGFβR2, p53, KRas, OGT, CASP5, COA-1,MAGE, SART or IL13Ralpha2.

It is also particularly preferred that such a complex comprises

-   -   one or more epitopes of EpCAM (such as the epitope according to        SEQ ID NO: 48) or functional sequence variants thereof;    -   one or more epitopes of MUC-1 (such as the epitope according to        SEQ ID NO: 50 and/or the epitope according to SEQ ID NO: 51) or        functional sequence variants thereof; and    -   one or more epitopes of CEA (such as the epitope according to        SEQ ID NO: 55 and/or the epitope according to SEQ ID NO: 56) or        functional sequence variants thereof.

Such a complex does preferably not comprise any epitope of HER-2,TOMM34, RNF 43, KOC1, VEGFR, βhCG, survivin, TGFβR2, p53, KRas, OGT,GASPS, COA-1, MAGE, SART or IL13Ralpha2.

It is also particularly preferred that such a complex comprises

-   -   one or more epitopes of EpCAM (such as the epitope according to        SEQ ID NO: 48) or functional sequence variants thereof; and    -   one or more epitopes of CEA (such as the epitope according to        SEQ ID NO: 55 and/or the epitope according to SEQ ID NO: 56) or        functional sequence variants thereof.

Such a complex does preferably not comprise any epitope of HER-2, MUC-1,TOMM34, RNF 43, KOC1, VEGFR, βhCG, survivin, TGFβR2, p53, KRas, OGT,CASP5, COA-1, MAGE, SART or IL13Ralpha2.

It is also particularly preferred that such a complex comprises

-   -   one or more epitopes of EpCAM (such as the epitope according to        SEQ ID NO: 48) or functional sequence variants thereof.

Such a complex does preferably not comprise any epitope of HER-2, MUC-1,TOMM34, RNF 43, KOC1, VEGFR, βhCG, survivin, CEA, TGFβR2, p53, KRas,OGT, CASP5, COA-1, MAGE, SART or IL13Ralpha2.

It is also particularly preferred that such a complex comprises

-   -   one or more epitopes of CEA (such as the epitope according to        SEQ ID NO: 55 and/or the epitope according to SEQ ID NO: 56) or        functional sequence variants thereof.

Such a complex does preferably not comprise any epitope of EpCAM, HER-2,MUC-1, TOMM34, RNF 43, KOC1, VEGFR, βhCG, survivin, TGFβR2, p53, KRas,OGT, GASPS, COA-1, MAGE, SART or IL13Ralpha2.

Preferably, such a complex comprises

-   -   one or more epitopes of EGFRvIII (such as the epitope according        to SEQ ID NO: 71) or functional sequence variants thereof;    -   one or more epitopes of EphA2 or functional sequence variants        thereof;    -   one or more epitopes of IL-13Rα2 (such as the epitope according        to SEQ ID NO: 62) or functional sequence variants thereof;    -   one or more epitopes of TRP-2 or functional sequence variants        thereof; and/or    -   one or more epitopes of brevican or functional sequence variants        thereof.

Such a complex does preferably not comprise any epitope of CMV, gp100,Her2/neu, survivin, hTert, MAGE-A1, MAGE-A3, YKL-40, neuroligin 4 orPTPRz1.

More preferably, such a complex comprises

-   -   one or more epitopes of EGFRvIII (such as the epitope according        to SEQ ID NO: 71) or functional sequence variants thereof;    -   one or more epitopes of EphA2 or functional sequence variants        thereof; one or more epitopes of IL-13Rα2 (such as the epitope        according to SEQ ID NO: 62) or functional sequence variants        thereof;    -   one or more epitopes of TRP-2 or functional sequence variants        thereof; and    -   one or more epitopes of brevican or functional sequence variants        thereof.

Such a complex does preferably not comprise any epitope of CMV, gp100,Her2/neu, survivin, hTert, MAGE-A1, MAGE-A3, YKL-40, neuroligin 4 orPTPRz1.

It is also more preferred that such a complex comprises

-   -   one or more epitopes of EphA2 or functional sequence variants        thereof;    -   one or more epitopes of IL-13Rα2 (such as the epitope according        to SEQ ID NO: 62) or functional sequence variants thereof;    -   one or more epitopes of TRP-2 or functional sequence variants        thereof; and    -   one or more epitopes of brevican or functional sequence variants        thereof.

Such a complex does preferably not comprise any epitope of EGFRvIII,CMV, gp100, Her2/neu, survivin, hTert, MAGE-A1, MAGE-A3, YKL-40,neuroligin 4 or PTPRz1.

It is also more preferred that such a complex comprises

-   -   one or more epitopes of EGFRvIII (such as the epitope according        to SEQ ID NO: 71) or functional sequence variants thereof;    -   one or more epitopes of IL-13Rα2 (such as the epitope according        to SEQ ID NO: 62) or functional sequence variants thereof;    -   one or more epitopes of TRP-2 or functional sequence variants        thereof; and    -   one or more epitopes of brevican or functional sequence variants        thereof.

Such a complex does preferably not comprise any epitope of EphA2, CMV,gp100, Her2/neu, survivin, hTert, MAGE-A1, MAGE-A3, YKL-40, neuroligin 4or PTPRz1.

It is also more preferred that such a complex comprises

-   -   one or more epitopes of EGFRvIII (such as the epitope according        to SEQ ID NO: 71) or functional sequence variants thereof;    -   one or more epitopes of EphA2 or functional sequence variants        thereof;    -   one or more epitopes of TRP-2 or functional sequence variants        thereof; and    -   one or more epitopes of brevican or functional sequence variants        thereof.

Such a complex does preferably not comprise any epitope of IL-13Ralpha2,CMV, gp100, Her2/neu, survivin, hTert, MAGE-A1, MAGE-A3, YKL-40,neuroligin 4 or PTPRz1.

It is also more preferred that such a complex comprises

-   -   one or more epitopes of EGFRvIII (such as the epitope according        to SEQ ID NO: 71) or functional sequence variants thereof;    -   one or more epitopes of EphA2 or functional sequence variants        thereof;    -   one or more epitopes of IL-13Rα2 (such as the epitope according        to SEQ ID NO: 62) or functional sequence variants thereof; and    -   one or more epitopes of brevican or functional sequence variants        thereof.

Such a complex does preferably not comprise any epitope of TRP-2, CMV,gp100, Her2/neu, survivin, hTert, MAGE-A1, MAGE-A3, YKL-40, neuroligin 4or PTPRz1.

It is also more preferred that such a complex comprises

-   -   one or more epitopes of EGFRvIII (such as the epitope according        to SEQ ID NO: 71) or functional sequence variants thereof;    -   one or more epitopes of EphA2 or functional sequence variants        thereof;    -   one or more epitopes of IL-13Rα2 (such as the epitope according        to SEQ ID NO: 62) or functional sequence variants thereof; and    -   one or more epitopes of TRP-2 or functional sequence variants        thereof.

Such a complex does preferably not comprise any epitope of brevican,CMV, gp100, Her2/neu, survivin, hTert, MAGE-A1, MAGE-A3, YKL-40,neuroligin 4 or PTPRz1.

It is also more preferred that such a complex comprises

-   -   one or more epitopes of EGFRvIII (such as the epitope according        to SEQ ID NO: 71) or functional sequence variants thereof;    -   one or more epitopes of EphA2 or functional sequence variants        thereof; and    -   one or more epitopes of IL-13Rα2 (such as the epitope according        to SEQ ID NO: 62) or functional sequence variants thereof.

Such a complex does preferably not comprise any epitope of TRP-2,brevican, CMV, gp100, Her2/neu, survivin, hTert, MAGE-A1, MAGE-A3,YKL-40, neuroligin 4 or PTPRz1.

It is also more preferred that such a complex comprises

-   -   one or more epitopes of EGFRvIII (such as the epitope according        to SEQ ID NO: 71) or functional sequence variants thereof;    -   one or more epitopes of EphA2 or functional sequence variants        thereof; and    -   one or more epitopes of TRP-2 or functional sequence variants        thereof.

Such a complex does preferably not comprise any epitope of brevican,IL-13Ralpha2, CMV, gp100, Her2/neu, survivin, hTert, MAGE-A1, MAGE-A3,YKL-40, neuroligin 4 or PTPRz1.

It is also more preferred that such a complex comprises

-   -   one or more epitopes of EGFRvIII (such as the epitope according        to SEQ ID NO: 71) or functional sequence variants thereof;    -   one or more epitopes of EphA2 or functional sequence variants        thereof; and    -   one or more epitopes of brevican or functional sequence variants        thereof.

Such a complex does preferably not comprise any epitope of IL-13Ralpha2,TRP-2, CMV, gp100, Her2/neu, survivin, hTert, MAGE-A1, MAGE-A3, YKL-40,neuroligin 4 or PTPRz1.

It is also more preferred that such a complex comprises

-   -   one or more epitopes of EGFRvIII (such as the epitope according        to SEQ ID NO: 71) or functional sequence variants thereof;    -   one or more epitopes of IL-13Rα2 (such as the epitope according        to SEQ ID NO: 62) or functional sequence variants thereof; and    -   one or more epitopes of TRP-2 or functional sequence variants        thereof.

Such a complex does preferably not comprise any epitope of EphA2,brevican, CMV, gp100, Her2/neu, survivin, hTert, MAGE-A1, MAGE-A3,YKL-40, neuroligin 4 or PTPRz1.

It is also more preferred that such a complex comprises

-   -   one or more epitopes of EGFRvIII (such as the epitope according        to SEQ ID NO: 71) or functional sequence variants thereof;    -   one or more epitopes of IL-13Rα2 (such as the epitope according        to SEQ ID NO: 62) or functional sequence variants thereof; and    -   one or more epitopes of brevican or functional sequence variants        thereof.

Such a complex does preferably not comprise any epitope of EphA2, TRP-2,CMV, gp100, Her2/neu, survivin, hTert, MAGE-A1, MAGE-A3, YKL-40,neuroligin 4 or PTPRz1.

It is also more preferred that such a complex comprises

-   -   one or more epitopes of EGFRvIII (such as the epitope according        to SEQ ID NO: 71) or functional sequence variants thereof;    -   one or more epitopes of TRP-2 or functional sequence variants        thereof; and    -   one or more epitopes of brevican or functional sequence variants        thereof.

Such a complex does preferably not comprise any epitope of EphA2,IL-13Ralpha2, CMV, gp100, Her2/neu, survivin, hTert, MAGE-A1, MAGE-A3,YKL-40, neuroligin 4 or PTPRz1.

It is also more preferred that such a complex comprises

-   -   one or more epitopes of EphA2 or functional sequence variants        thereof;    -   one or more epitopes of IL-13Rα2 (such as the epitope according        to SEQ ID NO: 62) or functional sequence variants thereof; and    -   one or more epitopes of TRP-2 or functional sequence variants        thereof.

Such a complex does preferably not comprise any epitope of EGFRvIII,brevican, CMV, gp100, Her2/neu, survivin, hTert, MAGE-A1, MAGE-A3,YKL-40, neuroligin 4 or PTPRz1.

It is also more preferred that such a complex comprises

-   -   one or more epitopes of EphA2 or functional sequence variants        thereof;    -   one or more epitopes of IL-13Rα2 (such as the epitope according        to SEQ ID NO: 62) or functional sequence variants thereof; and    -   one or more epitopes of brevican or functional sequence variants        thereof.

Such a complex does preferably not comprise any epitope of EGFRvIII,TRP-2, CMV, gp100, Her2/neu, survivin, hTert, MAGE-A1, MAGE-A3, YKL-40,neuroligin 4 or PTPRz1.

It is also more preferred that such a complex comprises

-   -   one or more epitopes of EphA2 or functional sequence variants        thereof;    -   one or more epitopes of TRP-2 or functional sequence variants        thereof; and    -   one or more epitopes of brevican or functional sequence variants        thereof.

Such a complex does preferably not comprise any epitope of EGFRvIII,IL-13Ralpha2, CMV, gp100, Her2/neu, survivin, hTert, MAGE-A1, MAGE-A3,YKL-40, neuroligin 4 or PTPRz1.

It is also more preferred that such a complex comprises

-   -   one or more epitopes of IL-13Rα2 (such as the epitope according        to SEQ ID NO: 62) or functional sequence variants thereof;    -   one or more epitopes of TRP-2 or functional sequence variants        thereof; and    -   one or more epitopes of brevican or functional sequence variants        thereof.

Such a complex does preferably not comprise any epitope of EGFRvIII,EphA2, CMV, gp100, Her2/neu, survivin, hTert, MAGE-A1, MAGE-A3, YKL-40,neuroligin 4 or PTPRz1.

It is also more preferred that such a complex comprises

-   -   one or more epitopes of EGFRvIII (such as the epitope according        to SEQ ID NO: 71) or functional sequence variants thereof; and    -   one or more epitopes of EphA2 or functional sequence variants        thereof.

Such a complex does preferably not comprise any epitope of IL-13Ralpha2,brevican, TRP-2, CMV, gp100, Her2/neu, survivin, hTert, MAGE-A1,MAGE-A3, YKL-40, neuroligin 4 or PTPRz1.

It is also more preferred that such a complex comprises

-   -   one or more epitopes of EGFRvIII (such as the epitope according        to SEQ ID NO: 71) or functional sequence variants thereof; and    -   one or more epitopes of IL-13Rα2 (such as the epitope according        to SEQ ID NO: 62) or functional sequence variants thereof.

Such a complex does preferably not comprise any epitope of EphA2,brevican, TRP-2, CMV, gp100, Her2/neu, survivin, hTert, MAGE-A1,MAGE-A3, YKL-40, neuroligin 4 or PTPRz1.

It is also more preferred that such a complex comprises

-   -   one or more epitopes of EGFRvIII (such as the epitope according        to SEQ ID NO: 71) or functional sequence variants thereof; and    -   one or more epitopes of TRP-2 or functional sequence variants        thereof; and

Such a complex does preferably not comprise any epitope of EphA2,IL-13Ralpha2, brevican, CMV, gp100, Her2/neu, survivin, hTert, MAGE-A1,MAGE-A3, YKL-40, neuroligin 4 or PTPRz1.

It is also more preferred that such a complex comprises

-   -   one or more epitopes of EGFRvIII (such as the epitope according        to SEQ ID NO: 71) or functional sequence variants thereof; and    -   one or more epitopes of brevican or functional sequence variants        thereof.

Such a complex does preferably not comprise any epitope of EphA2,IL-13Ralpha2, TRP-2, CMV, gp100, Her2/neu, survivin, hTert, MAGE-A1,MAGE-A3, YKL-40, neuroligin 4 or PTPRz1.

It is also more preferred that such a complex comprises

-   -   one or more epitopes of EphA2 or functional sequence variants        thereof; and    -   one or more epitopes of IL-13Rα2 (such as the epitope according        to SEQ ID NO: 62) or functional sequence variants thereof;

Such a complex does preferably not comprise any epitope of EGFRvIII,TRP-2, brevican, CMV, gp100, Her2/neu, survivin, hTert, MAGE-A1,MAGE-A3, YKL-40, neuroligin 4 or PTPRz1.

It is also more preferred that such a complex comprises

-   -   one or more epitopes of EphA2 or functional sequence variants        thereof; and    -   one or more epitopes of TRP-2 or functional sequence variants        thereof.

Such a complex does preferably not comprise any epitope of EGFRvIII,IL-13Ralpha2, brevican, CMV, gp100, Her2/neu, survivin, hTert, MAGE-A1,MAGE-A3, YKL-40, neuroligin 4 or PTPRz1.

It is also more preferred that such a complex comprises

-   -   one or more epitopes of EphA2 or functional sequence variants        thereof; and    -   one or more epitopes of brevican or functional sequence variants        thereof.

Such a complex does preferably not comprise any epitope of EGFRvIII,IL-13Ralpha2, TRP-2, CMV, gp100, Her2/neu, survivin, hTert, MAGE-A1,MAGE-A3, YKL-40, neuroligin 4 or PTPRz1.

It is also more preferred that such a complex comprises

-   -   one or more epitopes of IL-13Rα2 (such as the epitope according        to SEQ ID NO: 62) or functional sequence variants thereof; and    -   one or more epitopes of TRP-2 or functional sequence variants        thereof.

Such a complex does preferably not comprise any epitope of EGFRvIII,EphA2, brevican, CMV, gp100, Her2/neu, survivin, hTert, MAGE-A1,MAGE-A3, YKL-40, neuroligin 4 or PTPRz1.

It is also more preferred that such a complex comprises

-   -   one or more epitopes of IL-13Rα2 (such as the epitope according        to SEQ ID NO: 62) or functional sequence variants thereof; and    -   one or more epitopes of brevican or functional sequence variants        thereof.

Such a complex does preferably not comprise any epitope of EGFRvIII,EphA2, TRP-2, CMV, gp100, Her2/neu, survivin, hTert, MAGE-A1, MAGE-A3,YKL-40, neuroligin 4 or PTPRz1.

It is also more preferred that such a complex comprises

-   -   one or more epitopes of TRP-2 or functional sequence variants        thereof; and    -   one or more epitopes of brevican or functional sequence variants        thereof.

Such a complex does preferably not comprise any epitope of EGFRvIII,EphA2, IL-13Ralpha2, CMV, gp100, Her2/neu, survivin, hTert, MAGE-A1,MAGE-A3, YKL-40, neuroligin 4 or PTPRz1.

It is also more preferred that such a complex comprises

-   -   one or more epitopes of EGFRvIII (such as the epitope according        to SEQ ID NO: 71) or functional sequence variants thereof.

Such a complex does preferably not comprise any epitope of EphA2, TRP-2,brevican, IL-13Ralpha2, CMV, gp100, Her2/neu, survivin, hTert, MAGE-A1,MAGE-A3, YKL-40, neuroligin 4 or PTPRz1.

It is also more preferred that such a complex comprises

-   -   one or more epitopes of EphA2 or functional sequence variants        thereof.

Such a complex does preferably not comprise any epitope of EGFRvIII,IL-13Ralpha2, TRP-2, brevican, CMV, gp100, Her2/neu, survivin, hTert,MAGE-A1, MAGE-A3, YKL-40, neuroligin 4 or PTPRz1.

It is also more preferred that such a complex comprises

-   -   one or more epitopes of IL-13Rα2 (such as the epitope according        to SEQ ID NO: 62) or functional sequence variants thereof.

Such a complex does preferably not comprise any epitope of EGFRvIII,EphA2, TRP-2, brevican, CMV, gp100, Her2/neu, survivin, hTert, MAGE-A1,MAGE-A3, YKL-40, neuroligin 4 or PTPRz1.

It is also more preferred that such a complex comprises

-   -   one or more epitopes of TRP-2 or functional sequence variants        thereof.

Such a complex does preferably not comprise any epitope of EGFRvIII,EphA2, IL-13Ralpha2, brevican, CMV, gp100, Her2/neu, survivin, hTert,MAGE-A1, MAGE-A3, YKL-40, neuroligin 4 or PTPRz1.

It is also more preferred that such a complex comprises

-   -   one or more epitopes of brevican or functional sequence variants        thereof.

Such a complex does preferably not comprise any epitope of EGFRvIII,EphA2, IL-13Ralpha2, TRP-2, CMV, gp100, Her2/neu, survivin, hTert,MAGE-A1, MAGE-A3, YKL-40, neuroligin 4 or PTPRz1.

Component c)—TLR Peptide Agonist

In the complex according to the present invention, the TLR peptideagonist allows an increased targeting of the vaccine towards dendriticcells along with self-adjuvancity. Physical linkage of a TLR peptideagonist to the CPP and the at least one antigen or antigenic epitopeaccording to the present invention in the complex according to thepresent invention provides an enhanced immune response by simultaneousstimulation of antigen presenting cells, in particular dendritic cells,that internalize, metabolize and display antigen(s).

As used in the context of the present invention, a “TLR peptide agonist”is an agonist of a Toll-like receptor (TLR), i.e. it binds to a TLR andactivates the TLR, in particular to produce a biological response.Moreover, the TLR peptide agonist is a peptide, a polypeptide or aprotein as defined above. Preferably, the TLR peptide agonist comprisesfrom 10 to 150 amino acids, more preferably from 15 to 130 amino acids,even more preferably from 20 to 120 amino acids, particularly preferablyfrom 25 to 110 amino acids, and most preferably from 30 to 100 aminoacids.

Toll like receptors (TLRs) are transmembrane proteins that arecharacterized by extracellular, transmembrane, and cytosolic domains.The extracellular domains containing leucine-rich repeats (LRRs) withhorseshoe-like shapes are involved in recognition of common molecularpatterns derived from diverse microbes. Toll like receptors includeTLRs1-10. Compounds capable of activating TLR receptors andmodifications and derivatives thereof are well documented in the art.TLR1 may be activated by bacterial lipoproteins and acetylated formsthereof, TLR2 may in addition be activated by Gram positive bacterialglycolipids, LPS, LP A, LTA, fimbriae, outer membrane proteins, heatshock proteins from bacteria or from the host, and Mycobacteriallipoarabinomannans. TLR3 may be activated by dsRNA, in particular ofviral origin, or by the chemical compound poly(LC). TLR4 may beactivated by Gram negative LPS, LTA, Heat shock proteins from the hostor from bacterial origin, viral coat or envelope proteins, taxol orderivatives thereof, hyaluronan containing oligosaccharides andfibronectins. TLR5 may be activated with bacterial flagellae orflagellin. TLR6 may be activated by mycobacterial lipoproteins and groupB streptococcus heat labile soluble factor (GBS-F) or staphylococcusmodulins. TLR7 may be activated by imidazoquinolines. TLR9 may beactivated by unmethylated CpG DNA or chromatin—IgG complexes.

Preferably, the TLR peptide agonist comprised by the complex accordingto the present invention is an agonist of TLR1, 2, 4, 5, 6, and/or 10.TLRs are expressed either on the cell surface (TLR1, 2, 4, 5, 6, and 10)or on membranes of intracellular organelles, such as endosomes (TLR3, 4,7, 8, and 9). The natural ligands for the endosomal receptors turned outto be nucleic acid-based molecules (except for TLR4). The cellsurface-expressed TLR1, 2, 4, 5, 6, and 10 recognize molecular patternsof extracellular microbes (Monie, T. P., Bryant, C. E., et al. 2009:Activating immunity: Lessons from the TLRs and NLRB. Trends Biochem.Sci. 34(11), 553-561). TLRs are expressed on several cell types butvirtually all TLRs are expressed on DCs allowing these specialized cellsto sense all possible pathogens and danger signals.

However, TLR2, 4, and 5 are constitutively expressed at the surface ofDCs. Accordingly, the TLR peptide agonist comprised by the complexaccording to the present invention is more preferably a peptide agonistof TLR2, TLR4 and/or TLR5. Even more preferably, the TLR peptide agonistis a TLR2 peptide agonist and/or a TLR4 peptide agonist. Particularlypreferably, the TLR peptide agonist is a TLR4 peptide agonist. Mostpreferably, the TLR peptide agonist is one TLR peptide agonist, which isboth, a TLR2 and a TLR4 agonist. TLR2 can detect a wide variety ofligands derived from bacteria, viruses, parasites, and fungi. The ligandspecificity is often determined by the interaction of TLR2 with otherTLRs, such as TLR1, 6, or 10, or non-TLR molecules, such as dectin-1,CD14, or CD36. The formation of a heterodimer with TLR1 enables TLR2 toidentify triacyl lipoproteins or lipopeptides from (myco)bacterialorigin, such as Pam3CSK4 and peptidoglycan (PGA; Gay, N. J., andGangloff, M. (2007): Structure and function of Toll receptors and theirligands. Annu. Rev. Biochem. 76, 141-165; Spohn, R., Buwitt-Beckmann,U., et al. (2004): Synthetic lipopeptide adjuvants and Toll-likereceptor 2—Structure-activity relationships. Vaccine 22(19), 2494-2499).

Heterodimerization of TLR2 and 6 enables the detection of diacyllipopeptides and zymosan. Lipopolysaccharide (LPS) and its derivativesare ligands for TLR4 and flagellin for TLR5 (Bryant, C. E., Spring, D.R., et al. (2010). The molecular basis of the host response tolipopolysaccharide. Nat. Rev. Microbiol. 8(1), 8-14).

TLR2 interacts with a broad and structurally diverse range of ligands,including molecules expressed by microbes and fungi. Multiple TLR2agonists have been identified, including natural and syntheticlipopeptides (e.g. Mycoplasma fermentas macrophage-activatinglipopeptide (MALP-2)), peptidoglycans (PG such as those from S. aureus),lipopolysaccharides from various bacterial strains (LPS),polysaccharides (e.g. zymosan), glycosylphosphatidyl-inositol-anchoredstructures from gram positive bacteria (e.g. lipoteichoic acid (LTA) andlipo-arabinomannan from mycobacteria and lipomannas from M.tuberculosis). Certain viral determinants may also trigger via TLR2(Barbalat R, Lau L, Locksley R M, Barton G M. Toll-like receptor 2 oninflammatory monocytes induces type I interferon in response to viralbut not bacterial ligands. Nat Immunol. 2009: 10(11):1200-7). Bacteriallipopeptides are structural components of cell walls. They consist of anacylated s-glycerylcysteine moiety to which a peptide can be conjugatedvia the cysteine residue. Examples of TLR2 agonists, which are bacteriallipopeptides, include MALP-2 and it's synthetic analoguedi-palmitoyl-S-glyceryl cysteine (Pam₂Cys) or tri-palmitoyl-S-glycerylcysteine (Pam₃Cys).

A diversity of ligands interact with TLR4, including MonophosphorylLipid A from Salmonella minnesota R595 (MPLA), lipopolysaccharides(LPS), mannans (Candida albicans), glycoinositolphospholipids(Trypanosoma), viral envelope proteins (RSV and MMTV) and endogenousantigens including fibrinogen and heat-shock proteins. Such agonists ofTLR4 are for example described in Akira S, Uematsu S, Takeuchi O.Pathogen recognition and innate immunity. Cell. Feb. 24; 2006:124(4):783-801 or in Kumar H, Kawai T, Akira S. Toll-like receptors andinnate immunity. Biochem Biophys Res Commun. October 30; 2009388(4):621-5. LPS, which is found in the outer membrane of gram negativebacteria, is the most widely studied of the TLR4 ligands. SuitableLPS-derived TLR4 agonist peptides are described for example in WO2013/120073 (A1).

TLR5 is triggered by a region of the flagellin molecule expressed bynearly all motile bacteria. Thus, flagellin, or peptides or proteinsderived from flagellin and/or variants or fragments of flagellin arealso suitable as TLR peptide agonists comprised by the complex accordingto the present invention.

Examples of TLR peptide agonists thus include the TLR2 lipopeptideagonists MALP-2, Pam2Cys and Pam₃Cys or modifications thereof, differentforms of the TLR4 agonist LPS, e.g. N. meningitidis wild-type L3-LPS andmutant penta-acylated LpxL1-LPS, and the TLR5 agonist flagellin.

However, it is preferred that the TLR peptide agonist comprised by thecomplex according to the present invention is neither a lipopeptide nora lipoprotein, neither a glycopeptide nor a glycoprotein, morepreferably, the TLR peptide agonist comprised by the complex accordingto the present invention is a classical peptide, polypeptide or proteinas defined herein.

A preferred TLR2 peptide agonist is annexin II or an immunomodulatoryfragment thereof, which is described in detail in WO 2012/048190 A1 andUS patent application 13/0331546, in particular a TLR2 peptide agonistcomprising an amino acid sequence according to SEQ ID NO: 4 or SEQ IDNO: 7 of WO 2012/048190 A1 or fragments or variants thereof arepreferred.

Thereby, a TLR2 peptide agonist comprising or consisting of an aminoacid sequence according to SEQ ID NO: 15 or a sequence variant thereofas described above is particularly preferred as component c), i.e. asthe at least one TLR peptide agonist, comprised by the complex accordingto the present invention.

(TLR2 peptide agonist Anaxa) SEQ ID NO: 15STVHEILCKLSLEGDHSTPPSAYGSVKPYTNFDAE

Regarding TLR4, TLR peptides agonists are particularly preferred, whichcorrespond to motifs that bind to TLR4, in particular (i) peptidesmimicking the natural LPS ligand (RS01: Gln-Glu-Ile-Asn-Ser-Ser-Tyr andRS09: Ala-Pro-Pro-His-Ala-Leu-Ser) and (ii) Fibronectin derivedpeptides. The cellular glycoprotein Fibronectin (FN) has multipleisoforms generated from a single gene by alternative splicing of threeexons. One of these isoforms is the extra domain A (EDA), whichinteracts with TLR4.

Further suitable TLR peptide agonists comprise a fibronectin EDA domainor a fragment or variant thereof. Such suitable fibronectin EDA domainsor a fragments or variants thereof are disclosed in EP 1 913 954 B1, EP2 476 440 A1, US 2009/0220532 A1, and WO 2011/101332 A1. Thereby, a TLR4peptide agonist comprising or consisting of an amino acid sequenceaccording to SEQ ID NO: 45 or a sequence variant thereof as describedabove is particularly preferred as component c), i.e. as the at leastone TLR peptide agonist, comprised by the complex according to thepresent invention.

(TLR4 peptide agonist EDA) SEQ ID NO: 45NIDRPKGLAFTDVDVDSIKIAWESPQGQVSRYRVTYSSPEDGIRELFPAPDGEDDTAELQGLRPGSEYTVSVVALHDDMESQPLIGIQST

In addition, high-mobility group box 1 protein (HMGB1) and peptidefragments thereof are assumed to be TLR4 agonists. Such HMGB1-derivedpeptides are for example disclosed in US 2011/0236406 A1.

The complex according to the present invention comprises at least oneTLR peptide agonist, preferably the complex according to the presentinvention comprises more than one TLR peptide agonist, in particular 2,3, 4, 5, 6, 7, 8, 9, 10 or more TLR peptide agonists, more preferablythe complex according to the present invention comprises (at least) twoor three TLR peptide agonists, even more preferably the complexaccording to the present invention comprises (at least) four or five TLRpeptide agonists. If more than one TLR peptide agonist is comprised bythe complex according to the present invention it is understood thatsaid TLR peptide agonist is in particular also covalently linked in thecomplex according to the present invention, e.g. to another TLR peptideagonist and/or to a component a), i.e. a cell penetrating peptide,and/or to a component b), i.e. an antigen or antigenic epitope.

In a particularly preferred embodiment, the complex according to thepresent invention comprises one single TLR peptide agonist. Inparticularly, in this particularly preferred embodiment, the complexaccording to the present invention comprises one single TLR peptideagonist and no further component having TLR agonist properties exceptthe one single TLR peptide agonist as described.

The various TLR peptide agonists comprised by the complex according tothe present invention may be the same or different. Preferably, thevarious TLR peptide agonists comprised by the complex according to thepresent invention are different from each other.

Moreover, it is preferred that the more than one antigen or antigenicepitope, in particular 2, 3, 4, 5, 6, 7, 8, 9, 10 antigens or antigenicepitopes, or more TLR peptide agonists, in particular 2, 3, 4, 5, 6, 7,8, 9, 10 TLR agonists, are positioned consecutively in the complexaccording to the present invention. This means in particular that allTLR peptide agonists comprised by the complex are positioned in astretch, which is neither interrupted by component a), i.e. a cellpenetrating peptide, nor by component b), i.e. at least one antigen orantigenic epitope. Rather, component a) and component b) are positionedin the complex for example before or after such a stretch of all TLRpeptide agonists. However, the TLR peptide agonists positionedconsecutively in such a way may be linked to each other for example by aspacer or linker as described below, which is neither component a), i.e.a cell penetrating peptide, nor component b), i.e. at least one antigenor antigenic epitope.

Alternatively, however, the various TLR peptide agonists may also bepositioned in any other way in the complex according to the presentinvention, for example with component a) and/or component b) positionedin between two or more TLR peptide agonists, i.e. with one or more TLRpeptide agonist positioned between component a) and component b) (orvice versa) and, optionally, one or more TLR peptide agonists positionedat the respective other end of component a) and/or component b).

It is understood that a number of different TLR peptide agonistsactivating the same or different TLR receptors may be advantageouslycomprised by a single complex according to the present invention.Alternatively, a number of different TLR peptide agonists activating thesame or different TLR receptors may be distributed to subsets ofdifferent TLR peptide agonists activating the same or different TLRreceptors, which are comprised by different complexes according to thepresent invention, whereby such different complexes comprising differentsubsets may advantageously be administered simultaneously, e.g. in asingle vaccine, to a subject in need thereof.

Linkage of Components a), b), and c) in the Complex according to thePresent Invention

In the complex according to the present invention, components a), b) andc) are covalently linked, i.e. the linkage between two out of the threecomponents a), b), and c) of the complex according to the presentinvention is a covalent linkage. Preferably, two out of the threecomponents a), b), and c) of the complex according to the presentinvention are covalently linked to each other (i.e. the “first” and the“second” component), and the third component out of the three componentsa), b), and c) is covalently linked either to the first component out ofthe three components a), b), and c) or to the second component out ofthe three components a), b), and c). Thereby, preferably a linearmolecule is formed. However, it is also conceivable that each of thethree components a), b), and c) is covalently linked to both of theother components out of the three components a), b), and c).

A “covalent linkage” (also covalent bond), as used in the context of thepresent invention, refers to a chemical bond that involves the sharingof electron pairs between atoms. A “covalent linkage” (also covalentbond) in particular involves a stable balance of attractive andrepulsive forces between atoms when they share electrons. For manymolecules, the sharing of electrons allows each atom to attain theequivalent of a full outer shell, corresponding to a stable electronicconfiguration. Covalent bonding includes many kinds of interactions,including for example σ-bonding, π-bonding, metal-to-metal bonding,agostic interactions, and three-center two-electron bonds. Accordingly,the complex according to the present invention may also be referred toas “compound”, in particular it may be referred to as “molecule”.

Preferably, in the complex according to the present invention,components a), b), and c) are covalently linked by chemical coupling inany suitable manner known in the art, such as cross-linking methods.However, attention is drawn to the fact that many known chemicalcross-linking methods are non-specific, i.e., they do not direct thepoint of coupling to any particular site on the components a), b), andc). Thus, the use of non-specific cross-linking agents may attackfunctional sites or sterically block active sites, rendering the fusedcomponents of the complex according to the present inventionbiologically inactive. It is referred to the knowledge of the skilledartisan to block potentially reactive groups by using appropriateprotecting groups. Alternatively, the use of the powerful and versatileoxime and hydrazone ligation techniques, which are chemo-selectiveentities that can be applied for the cross-linking of components a), b),and c) may be employed. This linking technology is described e.g. byRose et al. (1994), JACS 116, 30.

Coupling specificity can be increased by direct chemical coupling to afunctional group found only once or a few times in components a), b),and/or c), which functional group is to be cross-linked to the anotherof the components a), b), and c). As an example, the cystein thiol groupmay be used, if just one cystein residue is present in a certaincomponent a), b), or c) of complex according to the present invention.Also, for example, if a certain component a), b), or c) contains nolysine residues, a cross-linking reagent specific for primary amineswill be selective for the amino terminus of the respective component.Alternatively, cross-linking may also be carried out via the side chainof a glutamic acid residue placed at the N-terminus of the peptide suchthat a amide bond can be generated through its side-chain. Therefore, itmay be advantageous to link a glutamic acid residue to the N-terminus ofa certain component a), b), or c). However, if a cysteine residue is tobe introduced into a certain component a), b), or c), introduction at ornear its N- or C-terminus is preferred. Conventional methods areavailable for such amino acid sequence alterations based onmodifications of certain component a), b), or c) by either adding one ormore additional amino acids, e.g. inter alia an cystein residue, to thetranslocation sequence or by substituting at least one residue of thetranslocation sequence(s) being comprised in the respective component.In case a cystein side chain is used for coupling purposes, a certaincomponent a), b), or c) has preferably one cystein residue. Any secondcystein residue should preferably be avoided and can, optionally, bereplaced when they occur in the respective component comprised by thecomplex according to the present invention. When a cysteine residue isreplaced in the original sequence of a certain component a), b), or c),it is typically desirable to minimize resulting changes in the peptidefolding of the respective component. Changes in folding are minimizedwhen the replacement is chemically and sterically similar to cysteine.Therefore, serine is preferred as a replacement for cystein.

Coupling of two out of the three components a), b), and c) can beaccomplished via a coupling or conjugating agent including standardpeptide synthesis coupling reagents such as HOBt, HBTU, DICI, TBTU.There are several intermolecular cross-linking agents which can beutilized, see for example, Means and Feeney, Chemical Modification ofProteins, Holden-Day, 1974, pp. 39-43. Among these reagents are, forexample, N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP) orN,N′-(1,3-phenylene)bismaleimide; N,N′-ethylene-bis-(iodoacetamide) orother such reagent having 6 to 11 carbon methylene bridges; and1,5-difluoro-2,4-dinitrobenzene. Other cross-linking agents useful forthis purpose include: p,p′-difluoro-m,m′-dinitrodiphenylsulfone;dimethyl adipimidate; phenol-1,4-disulfonylchloride;hexamethylenediisocyanate or di isothiocyanate, orazophenyl-p-diisocyanate; glutaraldehyde and disdiazobenzidine.Cross-linking agents may be homobifunctional, i.e., having twofunctional groups that undergo the same reaction. A preferredhomobifunctional cross-linking agent is bismaleimidohexane (BMH). BMHcontains two maleimide functional groups, which react specifically withsulfhydryl-containing compounds under mild conditions (pH 6.5-7.7). Thetwo maleimide groups are connected by a hydrocarbon chain. Therefore,BMH is useful for irreversible cross-linking of proteins (orpolypeptides) that contain cysteine residues. Cross-linking agents mayalso be heterobifunctional. Heterobifunctional cross-linking agents havetwo different functional groups, for example an amine-reactive group anda thiol-reactive group, that will cross-link two proteins having freeamines and thiols, respectively. Examples of heterobifunctionalcross-linking agents areSuccinimidyl-4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (SMCC),m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), and succinimide4-(p-maleimidophenyl)butyrate (SMPB), an extended chain analog of MBS.The succinimidyl group of these cross-linkers reacts with a primaryamine, and the thiol-reactive maleimide forms a covalent bond with thethiol of a cysteine residue. Because cross-linking agents often have lowsolubility in water, a hydrophilic moiety, such as a sulfonate group,may be added to the cross-linking agent to improve its water solubility.Sulfo-MBS and sulfo-SMCC are examples of cross-linking agents modifiedfor water solubility. Many cross-linking agents yield a conjugate thatis essentially non-cleavable under cellular conditions. Therefore, somecross-linking agents contain a covalent bond, such as a disulfide, thatis cleavable under cellular conditions. For example, Traut's reagent,dithiobis (succinimidylpropionate) (DSP), and N-succinimidyl3-(2-pyridyldithio)propionate (SPDP) are well-known cleavablecross-linkers. The use of a cleavable cross-linking agent permits thecell penetrating peptide, the at least one antigen or antigenic epitopeand the at least one TLR peptide agonist comprised by the complexaccording to the present invention to separate from each other afterdelivery into the target cell. For this purpose, direct disulfidelinkage may also be useful. Chemical cross-linking may also include theuse of spacer arms. Spacer arms provide intramolecular flexibility oradjust intramolecular distances between conjugated moieties and therebymay help preserve biological activity. A spacer arm may be in the formof a protein (or polypeptide) moiety that includes spacer amino acids,e.g. proline. Alternatively, a spacer arm may be part of thecross-linking agent, such as in “long-chain SPDP” (Pierce Chem. Co.,Rockford, Ill., cat. No. 21651 H). Numerous cross-linking agents,including the ones discussed above, are commercially available. Detailedinstructions for their use are readily available from the commercialsuppliers. More detailed information on protein cross-linking andconjugate preparation, which is useful in the context of linkage ofcomponents a), b), and c) comprised by the complex according to thepresent invention can be retrieved from: Wong, Chemistry of ProteinConjugation and Cross-Linking, CRC Press (1991).

Cross-linking agents for peptide or protein crosslinking include forexample (i) amine-to-amine crosslinkers, e.g. homobifunctionalamine-specific protein crosslinking reagents based on NHS-ester andimidoester reactive groups for selective conjugation of primary amines;available in short, long, cleavable, irreversible, membrane permeable,and cell surface varieties; (ii) sulfhydryl-to-carbohydratecrosslinkers, e.g. crosslinking reagents based on maleimide andhydrazide reactive groups for conjugation and formation of covalentcrosslinks; (iii) sulfhydryl-to-sulfhydryl crosslinkers, e.g.homobifunctional sulfhydryl-specific crosslinking reagents based onmaleimide or pyridyldithiol reactive groups for selective covalentconjugation of protein and peptide thiols (reduced cysteines) to formstable thioether bonds; (iv) photoreactive crosslinkers, e.g. arylazide, diazirine, and other photo-reactive (light-activated) chemicalheterobifunctional crosslinking reagents to conjugate proteins, nucleicacids and other molecular structures involved in receptor-ligandinteraction complexes via two-step activation; (v) amine-to-sulfhydrylcrosslinkers, e.g. heterobifunctional protein crosslinking reagents forconjugation between primary amine (lysine) and sulfhydryl (cysteine)groups of proteins and other molecules; available with different lengthsand types of spacer arms; and (vi) amine-to-amine crosslinkers, e.g.carboxyl-to-amine crosslinkers, e.g. Carbodiimide crosslinking reagents,DCC and EDC (EDAC), for conjugating carboxyl groups (glutamate,aspartate, C-termini) to primary amines (lysine, N-termini) and alsoN-hydroxysuccinimide (NHS) for stable activation of carboxylates foramine-conjugation.

Examples of crosslinkers in general, which can be used in the complexaccording to the present invention, includeN-(α-Maleimidoacetoxy)-succinimide ester,N-5-Azido-2-nitrobenzyloxy-succinimide, 1,4-Bis-Maleimidobutane,1,4-Bis-Maleimmidyl-2,3-dihydroxy-butane, Bis-Maleimidohexane,Bis-Maleimidoethane, N-(β-Maleimidopropionic acid)hydrazide*TFA,N-(β-Maleimidopropyloxy)succinimide ester,1,8-Bis-Maleimidodiethylene-glycol, 1,11-Bis-Maleimidotriethyleneglycol,Bis (sulfosuccinimidyl)suberate, Bis (sulfosuccinimidyl)glutarate-d0,Bis (sulfosuccinimidyl)2,2,4,4-glutarate-d4, Bis(sulfosuccinimidyl)suberate-d0, Bis(sulfosuccinimidyl)2,2,7,7-suberate-d4, Bis (NHS)PEG5, Bis (NHS)PEG9,Bis (2-[succinimidoxycarbonyloxy]ethyl)sulfone,N,N-Dicyclohexylcarbodiimide, 1-5-Difluoro-2,4-dinitrobenzene, Dimethyladipimidate*2HCl, Dimethyl pimelimidate*2HCl, Dimethylsuberimidate*2HCl, Disuccinimidyl glutarate,Dithiobis(succimidylpropionate) (Lomant's Reagent), Disuccinimidylsuberate, Disuccinimidyl tartarate, Dimethyl3,3′-dithiobispropionimidate*2HCl, Dithiobis-maleimidoethane,3,3′-Dithiobis (sulfosucci nimidylpropionate), 1-Ethyl-3-(3-dimethylaminopropyl) carbodi imide hydrochloride, Ethylene glycol bis (succinimidylsuccinate), N-ε-Maleimidocaproic acid, N-(ε-Maleimidocaproicacid)hydrazide, N-(ε-Maleimidocaproyloxy)succinimide ester,N-(γ-Maleimidobutyryloxy)succinimide ester, N-(κ-Maleimidoundecanoicacid)hydrazide, NHS-LC-Diazirine, Succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxy-(6-amidocaproate, Succinimidyl6-(3′-[2-pyridyldithio] propionamido)hexanoate, L-Photo-Leucine,L-Photo-Methionine, m-Maleimidobenzoyl-N-hydroxysuccinimide ester,4-(4-N-Maleimidophenyl)-butyric acid hydrazide*HCl,2-[N2-(4-Azido-2,3,5,6-tetrafluorobenzoyl)-N6-(6-biotinamidocaproyl)-L-lysinyl]ethylmethanethiosulfate,2-{N2-[N6-(4-Azido-2,3,5,6-tetrafluorobenzoyl)-N6-(6-biotinamidocaproyl)-L-lysinyl]}ethylmethanethiosulfate,N-Hydroxysuccinimide, N-hydroxysuccinimide ester ethane azide,N-hydroxysuccinimide ester tetraoxapentadecane azide,N-hydroxysuccinimide ester dodecaoxanonatriacontane azide,NHS-Phosphine, 3-(2-Pyridyldithio)propionylhydrazide,2-pyridyldithiol-tetraoxatetradecane-N-hydroxysuccinimide,2-pyridyldithiol-tetraoxaoctatriacontane-N-hydroxysuccinimide,N-(p-Maleimidophenyl)isocyanate, Succinimdyl3-(bromoacetamido)propionate, NHS-Diazirine, NHS-SS-Diazirine,N-succinimidyl iodoacetate, N-Succinimidyl(4-iodoacetyl)aminobenzoate,Succinimidyl 4-(N-maleimido-methyl)cyclohexane-1-carboxylate,NHS-PEG2-Maliemide, NHS-PEG4-Maliemide, NHS-PEG6-Maleimide,NHS-PEG8-Maliemide, NHS-PEG12-Maliemide, NHS-PEG24-Maleimide, Succinimidyl 4-(p-maleimido-phenyl)butyrate,Succinimidyl-6-(β-maleimidopropionamido)hexanoate,4-Succinimidyloxycarbonyl-methyl-α-(2-pyridyldithio)toluene,Succinimidyl-(4-psoralen-8-yloxy)butyrate, N-Succinimidyl3-(2-pyridyldithio)propionate, Ethylene glycol bis (sulfo-succinimidylsuccinate), N-(ε-Maleimidocaproyloxy)sulfosuccinimide ester,N-(γ-Maleimidobutryloxy)sulfosuccinimide ester,N-(κ-Maleimidoundecanoyloxy)sulfosuccinimide ester,Sulfo-NHS-LC-Diazirine, Sulfosuccinimidyl6-(3′-[2-pyridyldithio]propionamido)hexanoate,m-Maleimidobenzoyl-N-hydroxysulfosuccinimide ester,N-Hydroxysuccinimide, Sulfo-NHS-Phosphine, Sulfosuccinimidyl6-(4′-azido-2′-nitrophenylamino)hexanoate,Sulfo-NHS-(2-6-[Biotinamido]-2-(p-azidobezamido), Sulfo-NHS-Diazirine,Sulfo-NHS-SS-Diazirine, Sulfosucci nimidyl(4-iodo-acetyl)aminobenzoate,Sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate,Sulfosuccinimidyl 4-(p-maleimidophenyl)butyrate,Tris-(2-Maleimidoethyl)amine (Trifunctional), and Tris-(succimimidylaminotricetate) (Trifunctional).

The linkage between two out of the three components a), b), and c) ofthe complex according to the present invention may be directly orindirectly, i.e. two components directly adjoin or they may be linked byan additional component of the complex, e.g. a spacer or a linker.

A direct linkage may be realized preferably by an amide bridge, if thecomponents to be linked have reactive amino or carboxy groups. Morespecifically, if the components to be linked are peptides, polypeptidesor proteins, a peptide bond is preferred. Such a peptide bond may beformed using a chemical synthesis involving both components (anN-terminal end of one component and the C-terminal end of the othercomponent) to be linked, or may be formed directly via a proteinsynthesis of the entire peptide sequence of both components, whereinboth (protein or peptide) components are preferably synthesized in onestep. Such protein synthesis methods include e.g., without being limitedthereto, liquid phase peptide synthesis methods or solid peptidesynthesis methods, e.g. solid peptide synthesis methods according toMerrifield, t-Boc solid-phase peptide synthesis, Fmoc solid-phasepeptide synthesis, BOP(Benzotriazole-1-yl-oxy-tris-(dimethylamino)-phosphoniumhexafluorophosphate) based solid-phase peptide synthesis, etc.Alternatively, ester or ether linkages are preferred.

Moreover, in particular if the components to be linked are peptides,polypeptides or proteins, a linkage may occur via the side chains, e.g.by a disulfide bridge. Further components of other chemical nature, e.g.the at least one antigen or antigenic epitope if it is not of peptidicnature, may be likewise attached to the components of peptidic nature,e.g. the cell penetrating peptide, the at least one TLR peptide agonist,and the at least one antigen or antigenic epitope if it is of peptidicnature. The linkage via a side chain will preferably be based on sidechain amino, thiol or hydroxyl groups, e.g. via an amide or ester orether linkage. A linkage of a peptidic main chain with a peptidic sidechain of another component may also be via an isopeptide bond. Anisopeptide bond is an amide bond that is not present on the main chainof a protein. The bond forms between the carboxyl terminus of onepeptide or protein and the amino group of a lysine residue on another(target) peptide or protein.

The complex according to the present invention may optionally comprise aspacer or linker, which are non-immunologic moieties, which arepreferably cleavable, and which link component a) and b) and/orcomponent a) and c), and/or component b) and c), and/or link consecutiveantigens or antigenic epitopes, and/or link consecutive TLR peptideagonists, and/or link consecutive cell penetrating peptides, and/orwhich can be placed at the C-terminal part of components b) and/or c). Alinker or spacer may preferably provide further functionalities inaddition to linking of the components, and preferably being cleavable,more preferably naturally cleavable inside the target cell, e.g. byenzymatic cleavage. However, such further functionalities do inparticular not include any immunological functionalities. Examples offurther functionalities, in particular regarding linkers in fusionproteins, can be found in Chen X. et al., 2013: Fusion Protein Linkers:Property, Design and Functionality. Adv Drug Deliv Rev. 65(10):1357-1369, wherein for example also in vivo cleavable linkers aredisclosed. Moreover, Chen X. et al., 2013: Fusion Protein Linkers:Property, Design and Functionality. Adv Drug Deliv Rev. 65(10):1357-1369 also discloses various linkers, e.g. flexible linkers andrigid linkers, and linker designing tools and databases, which can beuseful in the complex according to the present invention or to design alinker to be used in the complex according to the present invention.

Said spacer may be peptidic or non-peptidic, preferably the spacer ispeptidic. Preferably, a peptidic spacer consists of about 1, 2, 3, 4, 5,6, 7, 8, 9, or 10 amino acids, more preferably of about 1, 2, 3, 4, or 5amino acids. The amino acid sequence of the peptidic spacer may beidentical to that of the N-terminal or C-terminal flanking region of anyof the components a), b), or c). Alternatively a peptidic spacer canconsist of non-natural amino acid sequences such as an amino acidsequence resulting from conservative amino acid substitutions of saidnatural flanking regions or sequences of known cleavage sites forproteases such as an enterokinase target site (amino acid sequence:DDDK, SEQ ID NO: 16), factor Xa target site (amino acid sequence: IEDGR,SEQ ID NO: 17), thrombin target site (amino acid sequence: LVPRGS, SEQID NO: 18), protease TEV target site (amino acid sequence: ENLYFQG, SEQID NO: 19), PreScission protease target site (amino acid sequenceLEVLFQGP, SEQ ID NO: 20), polycationic amino acids, e.g. poly K, furintarget site (amino acid sequence RX(R/K)R, SEQ ID NO: 21). In aparticular embodiment, the peptidic spacer does not contain any Cys (C)residues. In a preferred embodiment the linker sequence contains atleast 20%, more preferably at least 40% and even more preferably atleast 50% Gly or β-alanine residues, e.g. GlyGlyGlyGlyGly (SEQ ID NO:22), GlyGlyGlyGly (SEQ ID NO: 23), GlyGlyGly, CysGlyGly or GlyGlyCys,etc. Appropriate linker sequences can be easily selected and prepared bya person skilled in the art. They may be composed of D and/or L aminoacids. Further examples of a peptidic spacer include the amino acidsequences EQLE (SEQ ID NO: 24) or TEWT (SEQ ID NO: 25) or anyconservative substitutions thereof.

A non-peptidic spacer can include or may be an ester, a thioester, and adi-sulfide.

In particular, the complex according to the invention may comprise aspacer or linker, in particular a peptidic spacer, placed betweencomponent a) and b) and/or between component a) and c), and/or betweencomponent b) and c),. This peptidic spacer can be chosen by one skilledin the art so that it may be cut by the cell machinery once the complexcomprising the cell penetrating peptide and the cargo molecule has beeninternalized.

When the complex comprises several antigens or antigenic epitopes orwhen the complex comprises several TLR peptide agonists, it will beclear for one skilled in the art that each of the antigens or antigenicepitopes and/or each of the TLR peptide agonists comprised in thecomplex of the invention can be either directly linked to each other orlinked via spacers or linkers such as, e.g., a peptidic spacerconsisting of a few amino acids. Alternatively, when the complexaccording to the present invention comprises several antigens orantigenic epitopes or when the complex comprises several TLR peptideagonists, it is also possible that some antigens or antigenic epitopesand/or some TLR peptide agonists comprised by the complex of theinvention are directly linked to each other and some other antigens orantigenic epitopes and/or some other TLR peptide agonists are linked viaspacers or linkers such as a peptidic spacer consisting of a few aminoacids.

For example, two successive antigens or antigenic epitopes or twosuccessive TLR peptide agonists comprised in the complex of theinvention are linked to each other by spacers consisting of the naturalflanking regions of said antigens or antigenic epitopes or of said TLRpeptide agonists, respectively. For example, the spacer used to link afirst antigen/antigenic epitope or a first TLR peptide agonist to asecond antigen/antigenic epitope or to a second TLR peptide agonist,respectively, may consists of up to about 8 amino acids corresponding toup to about 4 amino acids of the N-terminal or C-terminal flankingregion of the first antigen/antigenic epitope or the first TLR peptideagonist, followed by up to about 4 amino acids of the N-terminal orC-terminal flanking region of the second antigen/antigenic epitope orthe second TLR peptide agonist. In an illustration of the presentinvention, the spacer used to link a first antigen/antigenic epitope ora first TLR peptide agonist (“antigen/epitope/TLR peptide agonist 1”) toa second epitope (“antigen/epitope/TLR peptide agonist 2”) consists ofabout 8 amino acids corresponding to any possible combination rangingfrom: 0 flanking amino acid of antigen/epitope/TLR peptide agonist 1 and8 flanking amino acids of antigen/epitope/TLR peptide agonist 2, to 8flanking amino acids of antigen/epitope/TLR peptide agonist 1 and 0flanking amino acid of antigen/epitope/TLR peptide agonist 2, i.e.including 1 flanking amino acid of antigen/epitope/TLR peptide agonist 1and 7 flanking amino acids of antigen/epitope/TLR peptide agonist 2, 2flanking amino acid of antigen/epitope/TLR peptide agonist 1 and 6flanking amino acids of antigen/epitope/TLR peptide agonist 2, 3flanking amino acid of antigen/epitope/TLR peptide agonist 1 and 5flanking amino acids of antigen/epitope/TLR peptide agonist 2, 4flanking amino acid of antigen/epitope/TLR peptide agonist 1 and 4flanking amino acids of antigen/epitope/TLR peptide agonist 2, 5flanking amino acid of antigen/epitope/TLR peptide agonist 1 and 3flanking amino acids of antigen/epitope/TLR peptide agonist 2, 6flanking amino acid of antigen/epitope/TLR peptide agonist 1 and 2flanking amino acids of antigen/epitope/TLR peptide agonist 2, 7flanking amino acid of antigen/epitope/TLR peptide agonist 1 and 1flanking amino acid of antigen/epitope/TLR peptide agonist 2, 8 flankingamino acid of antigen/epitope/TLR peptide agonist 1 and 0 flanking aminoacids of antigen/epitope/TLR peptide agonist 2. It will be understoodthat the total of 8 amino acids constituting a spacer linking twoconsecutive antigen/epitope/TLR peptide agonist is not an absolute valueand the spacer could also be composed of a total of, for instance, 3amino acids, 4 amino acids, 5 amino acids, 6 amino acids, 7 amino acids,9 amino acids or 10 amino acids. Similarly, equivalent combinations asmentioned above are also an illustration of the invention in thesituation where a spacer has less or more than 8 amino acids.

In another particular illustration of the present invention, the spacerused to link a first antigen/antigenic epitope or a first TLR peptideagonist (“antigen/epitope/TLR peptide agonist 1”) to a secondantigen/antigenic epitope or to a second TLR peptide agonist,respectively, (“antigen/epitopefTLR peptide agonist 2”) consists of e.g.1, 2, 3, 4, or 5 amino acids. More particularly, said spacer's aminoacid sequence can correspond to the 4 amino acids of the N-terminal orC-terminal flanking region of antigen/epitope/TLR peptide agonist 1 orantigen/epitope/TLR peptide agonist 2. A spacer as described above mayalso be placed at the C-terminal part of the last antigen/epitope/TLRpeptide agonist comprised in the complex according to the presentinvention.

The technics for linking two of the three components a), b), and c) arewell documented in the literature and can depend on the nature of the atleast one antigen or antigenic epitope. For instance, linkages betweentwo of the three components a), b), and c) can be achieved via cleavabledisulphide linkages through total stepwise solid-phase synthesis orsolution-phase or solid-phase fragment coupling, stable amide,thiazolidine, oxime and hydrazine linkage, disulphide linkage, stablethiomaleimide linkage, peptide bond (including peptide bonds betweenamino acids of a fusion protein), or electrostatic or hydrophobicinteractions.

Preferably, the at least one antigen or antigenic epitope comprised bythe complex according to the present invention as well as any optionalspacer or linker comprised by the complex according to the presentinvention are of peptidic nature. More preferably, all components of thecomplex according to the present invention, e.g. the cell penetratingpeptide, the at least one antigen or antigenic epitope, which is apeptide, polypeptide or protein, the at least one TLR peptide agonistand any optional peptidic linker or spacer are linked in the complexaccording to the present invention by a peptide bond. Most preferably,the complex according to the present invention is thus a peptide,polypeptide or protein, such as a fusion protein, e.g. a recombinantfusion protein.

In this context, a complex comprising or consisting of an amino acidsequence according to SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQID NO: 33, SEQ ID NO: 34, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39,SEQ ID NO: 40, SEQ ID NO: 41, SEQ ID NO: 46 or SEQ ID NO: 69 or acomplex comprising or consisting of an amino acid sequence sharing atleast 50%, at least 60%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 95% or at least 98% sequence identitywith any of SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 33,SEQ ID NO: 34, SEQ ID NO: 37, SEQ ID NO: 38, SEQ ID NO: 39, SEQ ID NO:40, SEQ ID NO: 41, SEQ ID NO: 46 or SEQ ID NO: 69 is preferred; acomplex comprising or consisting of an amino acid sequence according toSEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 33, SEQ ID NO: 37, SEQ ID NO:39, SEQ ID NO: 40, SEQ ID NO: 41 or SEQ ID NO: 69 or a complexcomprising or consisting of an amino acid sequence sharing at least 50%,at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 90%, at least 95% or at least 98% sequence identity with any ofSEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 33, SEQ ID NO: 37, SEQ ID NO:39, SEQ ID NO: 40, SEQ ID NO: 41 or SEQ ID NO: 69 is more preferred; acomplex comprising or consisting of an amino acid sequence according toSEQ ID NO: 28, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO:41 or SEQ ID NO: 69 or a complex comprising or consisting of an aminoacid sequence sharing at least 50%, at least 60%, at least 70%, at least75%, at least 80%, at least 85%, at least 90%, at least 95% or at least98% sequence identity with SEQ ID NO: 28, SEQ ID NO: 37, SEQ ID NO: 39,SEQ ID NO: 40, SEQ ID NO: 41 or SEQ ID NO: 69 is even more preferred;and a complex comprising or consisting of an amino acid sequenceaccording to SEQ ID NO: 28, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 41or SEQ ID NO: 69 or a complex comprising or consisting of an amino acidsequence sharing at least 50%, at least 60%, at least 70%, at least 75%,at least 80%, at least 85%, at least 90%, at least 95% or at least 98%sequence identity with SEQ ID NO: 28, SEQ ID NO: 39, SEQ ID NO: 40, SEQID NO: 41 or SEQ ID NO: 69 is particularly preferred.

SEQ ID NO: 26: MHHHHHHNID RPKGLAFTDV DVDSIKIAWE SPQGQVSRYR VTYSSPEDGIRELFPAPDGEDDTAELQGLR PGSEYTVSVV ALHDDMESQP LIGIQSTKRY KNRVASRKSRAKFKQLLQHY REVAAAKSSE NDRLRLLLKE SLKISQAVHA AHAEINEAGR EVVGVGALKVPRNQDWLGVP RFAKFASFEA QGALANIAVD KANLDVEQLE SIINFEKLTE WTGS SEQ ID NO:27: MHHHHHHSTV HEILCKLSLE GDHSTPPSAY GSVKPYTNFD AEKRYKNRVA SRKSRAKFKQLLQHYREVAA AKSSENDRLR LLLKESLKIS QAVHAAHAEI NEAGREVVGV GALKVPRNQDWLGVPRFAKF ASFEAQGALA NIAVDKANLD VEQLESIINF EKLTEWTGS SEQ ID NO: 28:MHHHHHHKRYKNRVA SRKSRAKFKQ LLQHYREVAA AKSSENDRLR LLLKESLKIS QAVHAAHAEINEAGREVVGV GALKVPRNQD WLGVPRFAKF ASFEAQGALA NIAVDKANLD VEQLESIINFEKLTEWTGSS TVHEILCKLS LEGDHSTPPS AYGSVKPYTN FDAE SEQ ID NO: 33:MHHHHHHKRY KNRVASRKSR AKFKQLLQHY REVAAAKESL KISQAVHAAH AEINEAGREVVGVGALKVPR NQDWLGVPRF AKFASFEAQG ALANIAVDKA NLDVEQLESI INFEKLTEWTGSSTVHEILC KLSLEGDHST PPSAYGSVKP YTNFDAE SEQ ID NO: 34: MHHHHHHREVAAAKSSENDR LRLLLKESLK ISQAVHAAHA EINEAGREVV GVGALKVPRN QDWLGVPRFAKFASFEAQGA LANIAVDKAN LDVEQLESII NFEKLTEWTG SSTVHEILCK LSLEGDHSTPPSAYGSVKPY TNFDAE SEQ ID NO: 37: MHHHHHHNID RPKGLAFTDV DVDSIKIAWESPQGQVSRYR VTYSSPEDGI RELFPAPDGE DDTAELQGLR PGSEYTVSVV ALHDDMESQPLIGIQSTKRY KNRVASRKSR AKFKQLLQHY REVAAAKESL KISQAVHAAH AEINEAGREVVGVGALKVPR NQDWLGVPRF AKFASFEAQG ALANIAVDKA NLDVEQLESI INFEKLTEWT GS SEQID NO: 38: MHHHHHHNID RPKGLAFTDV DVDSIKIAWE SPQGQVSRYR VTYSSPEDGIRELFPAPDGE DDTAELQGLR PGSEYTVSVV ALHDDMESQP LIGIQSTREV AAAKSSENDRLRLLLKESLK ISQAVHAAHA EINEAGREVV GVGALKVPRN QDWLGVPRFA KFASFEAQGALANIAVDKAN LDVEQLESII NFEKLTEWTG S SEQ ID NO: 39:KRYKNRVASRKSRAKFKQLLQHYREVAAAKSSENDRLRLLLKVTYHSPSYAYHQFERRAILNRLVQFIKDRISVVQALVLTSTVHEILCKLSLEGDHSTPPSAYGSVKPYTN FDAE SEQ ID NO: 40:KRYKNRVASRKSRAKFKQLLQHYREVAAAKSSENDRLRLLLKNYRIATFKNWPFLEDCAMEELTVSEFLKLDRQRSTVHEILCKLSLEGDHSTPPSAYGSVKPYTNFDAE SEQ ID NO: 41:KRYKNRVASRKSRAKFKQLLQHYREVAAAKSSENDRLRLLLKHLELASMTNMELMSSIVSTVHEILCKLSLEGDHSTPPSAYGSVKPYTNFDAE SEQ ID NO: 46:RKKRRQRRRRVKRISQAVHAAHAEINEAGRRVKRKVPRNQDWLRVKRASFEAQGALANIAVDKARVKRSIINFEKLRVKRSTVHEILCKLSLEGDHSTPPSAYGSVKPYTNFDAE SEQ ID NO: 69:KRYKNRVASRKSRAKFKQLLQHYREVAAAKSSENDRLRLLLKLFRAAQLANDVVLQIMEHLELASMTNMELMSSIVVISASIIVFNLLELEGSTVHEILCKLSLEGDHSTPPSAYGSVKPYTNFDAE

Arrangement of Components a), b), and c) in the Complex according to thePresent Invention

The components a), b), and c) may be arranged in the complex accordingto the present invention in any way.

In particular if more than one cell penetrating peptide and/or more thanone antigen or antigenic epitope and/or more than one TLR peptideagonist are comprised by the complex according to the present invention,the more than one cell penetrating peptide may be positioned in anon-consecutive manner, i.e. at least one antigen or antigenic epitope(component b)) and/or at least one TLR peptide agonist (component c))may interrupt a stretch of consecutively positioned cell penetratingpeptides and/or the cell penetrating peptides may be positioned withcomponent b) and/or with component c) in an alternating manner.Similarly, the more than one antigen or antigenic epitope may bepositioned in a non-consecutive manner, i.e. at least one cellpenetrating peptide (component a)) and/or at least one TLR peptideagonist (component c)) may interrupt a stretch of consecutivelypositioned antigens or antigenic epitopes and/or the antigens orantigenic epitopes may be positioned with component a) and/or withcomponent c) in an alternating manner. Similarly, the more than one TLRpeptide agonist may be positioned in a non-consecutive manner, i.e. atleast one cell penetrating peptide (component a)) and/or at least oneantigen or antigenic epitope (component b)) may interrupt a stretch ofconsecutively positioned TLR peptide agonists and/or the TLR peptideagonists may be positioned with component a) and/or with component b) inan alternating manner.

However, it is preferred that the more than one cell penetrating peptideis positioned in the complex according to the present invention in aconsecutive manner and/or the more than one antigen or antigenic epitopeis positioned in the complex according to the present invention in aconsecutive manner and/or the more than one TLR peptide agonist ispositioned in the complex according to the present invention in aconsecutive manner. This means in particular that all single units of acertain component, i.e. all cell penetrating peptides, all antigens orantigenic epitopes or all TLR peptide agonists, which are comprised bythe complex are positioned in a stretch, which is not interrupted by anyof the other two components. Rather, the other two components arepositioned in the complex for example before or after such a stretch ofall single units of said certain component. However, the single units ofsaid certain component positioned consecutively in such a way may belinked to each other for example by a spacer or linker as describedherein, which is not of the other two components.

It is particularly preferred that each of the components a), b), and c)is positioned in a consecutive manner.

Structurally each component a), b), and c) typically comprises a singlemain chain and at least one side chain. The term “main chain” (also“backbone chain”), as used in the context of the present invention,refers to the main continuous chain of covalently bond atoms in amolecule. For example, in peptides, polypeptides and proteins, the mainchain (backbone) typically comprises alpha-carbon atoms and nitrogenatoms of the constituent amino acids linked by the peptide bond. Thebackbone does not include the side chains. The term “side chain” (also“pendant chain”), as used in the context of the present invention,refers to a chemical group that is attached to a core part of themolecule called “main chain” or backbone. For example, in peptides,polypeptides and proteins, the side chains typically represent the(main) parts of the constituent amino acids, which are attached to thealpha-carbon atoms of the backbone.

In the complex according to the present invention, the components a),b), and c) may be covalently linked via a linker or spacer as describedherein or they may be directly covalently linked. Independently ofwhether a spacer or linker is used for covalent linkage or not, thereare in principle four options of how two of the three components arelinked to each other in the complex according to the present invention,namely:

-   -   (i) via main-chain/main-chain linkage,    -   (ii) via main-chain/side-chain linkage,    -   (iii) via side-chain/main-chain linkage or    -   (iv) via side-chain/side chain linkage.

Preferably, all three components a), b), and c) are linked viamain-chain/main-chain linkage, thus resulting in particular in a mainchain of the complex according to the present invention, which comprisesthe main chain of one or more cell penetrating peptide(s), the mainchain of one or more antigen(s) or antigenic epitope(s), and the mainchain of one or more TLR peptide agonist(s). In other words, the mainchain of one or more cell penetrating peptide(s), the main chain of oneor more antigen(s) or antigenic epitope(s), and the main chain of one ormore TLR peptide agonist(s) constitute the main chain of the complexaccording to the present invention, optionally together with furthercomponents, for example linker(s), spacer(s), etc. Accordingly, thefollowing arrangements of the components a), b), and c) are preferred,in particular if the at least one antigen or antigenic epitope is apeptide, polypeptide or protein, whereby said preferred arrangements areshown below in N-terminus→C-terminus direction of the main chain of thecomplex and wherein all three components a), b), and c) are linked viamain-chain/main-chain linkage and may be optionally linked by a linker,a spacer or another additional component:

-   -   (α) component a) (cell penetrating peptide)—component b) (at        least one antigen or antigenic epitope)—component c) (at least        one TLR peptide agonist);    -   (β) component c) (at least one TLR peptide agonist)—component a)        (cell penetrating peptide)—component b) (at least one antigen or        antigenic epitope);    -   (γ) component a) (cell penetrating peptide)—component c) (at        least one TLR peptide agonist)—component b) (at least one        antigen or antigenic epitope);    -   (δ) component c) (at least one TLR peptide agonist)—component b)        (at least one antigen or antigenic epitope)—component a) (cell        penetrating peptide);    -   (ε) component b) (at least one antigen or antigenic        epitope)—component a) (cell penetrating peptide)—component c)        (at least one TLR peptide agonist); or    -   (ζ) component b) (at least one antigen or antigenic        epitope)—component c) (at least one TLR peptide        agonist)—component a) (cell penetrating peptide).

In particular if all three components a), b), and c) are linked viamain-chain/main-chain linkage, it is preferred that the at least oneantigen or antigenic epitope is positioned C-terminally of the cellpenetrating peptide, whereby the cell penetrating peptide and the atleast one antigen or antigenic epitope are optionally linked by afurther component, e.g. a linker, a spacer, or by the at least one TLRpeptide agonist. Accordingly, this corresponds to the arrangements (α),(β), and (γ) from the arrangements shown above, i.e. from the abovearrangements arrangements (α), (β), and (γ) are more preferred.

Even more preferably, the at least one antigen or antigenic epitope ispositioned C-terminally of the cell penetrating peptide, whereby thecell penetrating peptide and the at least one antigen or antigenicepitope are optionally linked by a further component, e.g. a linker, aspacer, but not by the at least one TLR peptide agonist. Accordingly,this corresponds to the arrangements (α) and (β) from the arrangementsshown above, i.e. from the above arrangements arrangements (α) and (β)are even more preferred. Particularly preferably, the complex accordingto the present invention is a recombinant polypeptide or a recombinantprotein and the components a) to c) are positioned inN-terminus→C-terminus direction of the main chain of said complex in theorder:

-   (α) component a)—component b)—component c); or-   (β) component c)—component a)—component b),

wherein the components may be linked by a further component, inparticular by a linker or a spacer.

Particularly preferred is arrangement (α), wherein the at least one TLRagonist comprises or consists of at least one TLR2 agonist, for example:

-   (α1) component a) (cell penetrating peptide)—component b) (at least    one antigen or antigenic epitope)—one or more TLR2 peptide agonist,    e.g. 1, 2, 3, 4, or 5 TLR2 peptide agonist(s);-   (α2) component a) (cell penetrating peptide)—component b) (at least    one antigen or antigenic epitope)—one or more TLR2 peptide agonist,    e.g. 1, 2, 3, 4, or 5 TLR2 peptide agonist(s), one or more TLR4    peptide agonist, e.g. 1, 2, 3, 4, or 5 TLR4 peptide agonist(s) and    one or more TLR5 peptide agonist, e.g. 1, 2, 3, 4, or 5 TLR5 peptide    agonist(s);-   (α3) component a) (cell penetrating peptide)—component b) (at least    one antigen or antigenic epitope)—one or more TLR2 peptide agonist,    e.g. 1, 2, 3, 4, or 5 TLR2 peptide agonist(s) and one or more TLR4    peptide agonist, e.g. 1, 2, 3, 4, or 5 TLR4 peptide agonist(s); or-   (α4) component a) (cell penetrating peptide)—component b) (at least    one antigen or antigenic epitope)—one or more TLR2 peptide agonist,    e.g. 1, 2, 3, 4, or 5 TLR2 peptide agonist(s) and one or more TLR5    peptide agonist, e.g. 1, 2, 3, 4, or 5 TLR5 peptide agonist(s).

Alternatively, in such an arrangement comprising a TLR2 peptide agonist,additional TLR peptide agonists may also be arranged at other positionsin the complex, for example:

-   (α5) one or more TLR4 peptide agonist, e.g. 1, 2, 3, 4, or 5 TLR4    peptide agonist(s)—component a) (cell penetrating    peptide)—component b) (at least one antigen or antigenic    epitope)—one or more TLR2 peptide agonist, e.g. 1, 2, 3, 4, or 5    TLR2 peptide agonist(s);-   (α6) one or more TLR5 peptide agonist, e.g. 1, 2, 3, 4, or 5 TLR5    peptide agonist(s)—component a) (cell penetrating    peptide)—component b) (at least one antigen or antigenic    epitope)—one or more TLR2 peptide agonist, e.g. 1, 2, 3, 4, or 5    TLR2 peptide agonist(s); or-   (α7) one or more TLR4 peptide agonist, e.g. 1, 2, 3, 4, or 5 TLR4    peptide agonist(s) and one or more TLR5 peptide agonist, e.g. 1, 2,    3, 4, or 5 TLR5 peptide agonist(s)—component a) (cell penetrating    peptide)—component b) (at least one antigen or antigenic    epitope)—one or more TLR2 peptide agonist, e.g. 1, 2, 3, 4, or 5    TLR2 peptide agonist(s).

Particularly preferred is arrangement (β), wherein the at least one TLRagonist comprises or consists of at least one TLR4 agonist, for example:

-   (β1) one or more TLR4 peptide agonist, e.g. 1, 2, 3, 4, or 5 TLR4    peptide agonist(s)—component a) (cell penetrating    peptide)—component b) (at least one antigen or antigenic epitope);-   (β2) one or more TLR4 peptide agonist, e.g. 1, 2, 3, 4, or 5 TLR4    peptide agonist(s), one or more TLR2 peptide agonist, e.g. 1, 2, 3,    4, or 5 TLR2 peptide agonist(s) and one or more TLR5 peptide    agonist, e.g. 1, 2, 3, 4, or 5 TLR5 peptide agonist(s)—component a)    (cell penetrating peptide)—component b) (at least one antigen or    antigenic epitope);-   (β3) one or more TLR4 peptide agonist, e.g. 1, 2, 3, 4, or 5 TLR4    peptide agonist(s) and one or more TLR2 peptide agonist, e.g. 1, 2,    3, 4, or 5 TLR2 peptide agonist(s)—component a) (cell penetrating    peptide)—component b) (at least one antigen or antigenic epitope);    or-   (β4) one or more TLR4 peptide agonist, e.g. 1, 2, 3, 4, or 5 TLR4    peptide agonist(s) and one or more TLR5 peptide agonist, e.g. 1, 2,    3, 4, or 5 TLR5 peptide agonist(s)—component a) (cell penetrating    peptide)—component b) (at least one antigen or antigenic epitope).

Alternatively, in such an arrangement comprising a TLR4 peptide agonist,additional TLR peptide agonists may also be arranged at other positionsin the complex, for example:

-   (β5) one or more TLR4 peptide agonist, e.g. 1, 2, 3, 4, or 5 TLR4    peptide agonist(s)—component a) (cell penetrating    peptide)—component b) (at least one antigen or antigenic    epitope)—one or more TLR2 peptide agonist, e.g. 1, 2, 3, 4, or 5    TLR2 peptide agonist(s);-   (β6) one or more TLR4 peptide agonist, e.g. 1, 2, 3, 4, or 5 TLR4    peptide agonist(s)—component a) (cell penetrating    peptide)—component b) (at least one antigen or antigenic    epitope)—one or more TLR5 peptide agonist, e.g. 1, 2, 3, 4, or 5    TLR5 peptide agonist(s); or-   (β7) one or more TLR4 peptide agonist, e.g. 1, 2, 3, 4, or 5 TLR4    peptide agonist(s)—component a) (cell penetrating    peptide)—component b) (at least one antigen or antigenic    epitope)—one or more TLR2 peptide agonist, e.g. 1, 2, 3, 4, or 5    TLR2 peptide agonist(s) and one or more TLR5 peptide agonist, e.g.    1, 2, 3, 4, or 5 TLR5 peptide agonist(s).

Alternatively, only two of the three components a), b), and c) arelinked via main-chain/main-chain linkage in the complex according to thepresent invention.

For example components a) and b) are linked via main-chain/main-chainlinkage, resulting thus in the following arrangements of the componentsa) and b) in the complex, shown in N-terminus→C-terminus direction ofthe main chain of the complex, whereby the components a) and b) may beoptionally linked by a further component, e.g. a linker, a spacer etc.:

-   -   (1) cell penetrating peptide (a)—antigen/antigenic epitope (b);        or    -   (2) antigen/antigenic epitope (b)—cell penetrating peptide (a).

In such a case, component c), i.e. the at least one TLR peptide agonist,may then be arranged via main-chain/side-chain linkage, viaside-chain/main-chain linkage or via side-chain/side chain linkage toeither the cell penetrating peptide (a) or to the antigen/antigenicepitope (b) or, if present, to an additional component like a spacer orlinker, which may be, for example, positioned between the cellpenetrating peptide (a) and the antigen/antigenic epitope (b). Thisincludes the following arrangements:

-   -   (i) component c) may be linked—optionally via a spacer or a        linker—via main-chain/side-chain linkage to component a), i.e.        the main chain of the at least one TLR peptide agonist is        covalently linked—optionally via a spacer or a linker—to the        side chain of the cell penetrating peptide;    -   (ii) component c) may be linked—optionally via a spacer or a        linker—via side-chain/main-chain linkage to component a), i.e.        the side chain of the at least one TLR peptide agonist is        covalently linked—optionally via a spacer or a linker—to the        main chain of the cell penetrating peptide;    -   (iii) component c) may be linked—optionally via a spacer or a        linker—via side-chain/side-chain linkage to component a), i.e.        the side chain of the at least one TLR peptide agonist is        covalently linked—optionally via a spacer or a linker—to the        side chain of the cell penetrating peptide;    -   (iv) component c) may be linked—optionally via a spacer or a        linker—via main-chain/side-chain linkage to component b), i.e.        the main chain of the at least one TLR peptide agonist is        covalently linked—optionally via a spacer or a linker—to the        side chain of the at least one antigen or antigenic epitope;    -   (v) component c) may be linked—optionally via a spacer or a        linker—via side-chain/main-chain linkage to component b), i.e.        the side chain of the at least one TLR peptide agonist is        covalently linked—optionally via a spacer or a linker—to the        main chain of the at least one antigen or antigenic epitope;    -   (vi) component c) may be linked—optionally via a spacer or a        linker—via side-chain/side-chain linkage to component b), i.e.        the side chain of the at least one TLR peptide agonist is        covalently linked—optionally via a spacer or a linker—to the        side chain of the at least one antigen or antigenic epitope;    -   (vii )component c) may be linked—optionally via a spacer or a        linker—via main-chain/side-chain linkage to a linker or a spacer        positioned between component a) and component b), i.e. the main        chain of the at least one TLR peptide agonist is covalently        linked—optionally via a spacer or a linker—to the side chain of        a linker or a spacer positioned between component a) and        component b);    -   (viii) component c) may be linked—optionally via a spacer or a        linker—via side-chain/main-chain linkage to a linker or a spacer        positioned between component a) and component b), i.e. the side        chain of the at least one TLR peptide agonist is covalently        linked—optionally via a spacer or a linker—to the main chain of        a linker or a spacer positioned between component a) and        component b); or    -   (ix) component c) may be linked—optionally via a spacer or a        linker—via side-chain/side-chain linkage to a linker or a spacer        positioned between component a) and component b), i.e. the side        chain of the at least one TLR peptide agonist is covalently        linked—optionally via a spacer or a linker—to the side chain of        a linker or a spacer positioned between component a) and        component b).

For example components b) and c) are linked via main-chain/main-chainlinkage, resulting thus in the following arrangements of the componentsb) and c) in the complex, shown in N-terminus→C-terminus direction ofthe main chain of the complex, whereby the components b) and c) may beoptionally linked by a further component, e.g. a linker, a spacer etc.:

-   -   (3) antigen/antigenic epitope (b)—TLR peptide agonist (c); or    -   (4) TLR peptide agonist (c)—antigen/antigenic epitope (b).

In such a case, component a), i.e. the cell penetrating peptide, maythen be arranged via main-chain/side-chain linkage, viaside-chain/main-chain linkage or via side-chain/side chain linkage toeither the antigen/antigenic epitope (b) or to the TLR peptide agonist(c) or, if present, to an additional component like a spacer or linker,which may be, for example, positioned between the antigen/antigenicepitope (b) and the TLR peptide agonist (c). This includes the followingarrangements:

-   -   (x) component a) may be linked—optionally via a spacer or a        linker—via main-chain/side-chain linkage to component b), i.e.        the main chain of the cell penetrating peptide is covalently        linked—optionally via a spacer or a linker—to the side chain of        the at least one antigen or antigenic epitope;    -   (xi) component a) may be linked—optionally via a spacer or a        linker—via side-chain/main-chain linkage to component b), i.e.        the side chain of the cell penetrating peptide is covalently        linked—optionally via a spacer or a linker—to the main chain of        the at least one antigen or antigenic epitope;    -   (xii) component a) may be linked—optionally via a spacer or a        linker—via side-chain/side-chain linkage to component b), i.e.        the side chain of the cell penetrating peptide is covalently        linked—optionally via a spacer or a linker—to the side chain of        the at least one antigen or antigenic epitope;    -   (xiii) component a) may be linked—optionally via a spacer or a        linker—via main-chain/side-chain linkage to component c), i.e.        the main chain of the cell penetrating peptide is covalently        linked—optionally via a spacer or a linker—to the side chain of        the at least one TLR peptide agonist;    -   (xiv) component a) may be linked—optionally via a spacer or a        linker—via side-chain/main-chain linkage to component c), i.e.        the side chain the cell penetrating peptide is covalently        linked—optionally via a spacer or a linker—to the main chain of        the at least one TLR peptide agonist;    -   (xv)component a) may be linked—optionally via a spacer or a        linker—via side-chain/side-chain linkage to component c), i.e.        the side chain of the cell penetrating peptide is covalently        linked—optionally via a spacer or a linker—to the side chain of        the at least one TLR peptide agonist;    -   (xvi) component a) may be linked—optionally via a spacer or a        linker—via main-chain/side-chain linkage to a linker or a spacer        positioned between component b) and component c), i.e. the main        chain of the cell penetrating peptide is covalently        linked—optionally via a spacer or a linker—to the side chain of        a linker or a spacer positioned between component b) and        component c);    -   (xvii) component a) may be linked—optionally via a spacer or a        linker—via side-chain/main-chain linkage to a linker or a spacer        positioned between component b) and component c), i.e. the side        chain of the cell penetrating peptide is covalently        linked—optionally via a spacer or a linker—to the main chain of        a linker or a spacer positioned between component b) and        component c); or    -   (xviii) component a) may be linked—optionally via a spacer or a        linker—via side-chain/side-chain linkage to a linker or a spacer        positioned between component b) and component c), i.e. the side        chain of the cell penetrating peptide is covalently        linked—optionally via a spacer or a linker—to the side chain of        a linker or a spacer positioned between component b) and        component c).

For example components a) and c) are linked via main-chain/main-chainlinkage, resulting thus in the following arrangements of the componentsa) and b) in the complex, shown in N-terminus→C-terminus direction ofthe main chain of the complex, whereby the components a) and c) may beoptionally linked by a further component, e.g. a linker, a spacer etc.:

-   -   (5) cell penetrating peptide (a)—TLR peptide agonist (c); or    -   (6) TLR peptide agonist (c)—cell penetrating peptide (a).

In such a case, component b), i.e. the at least one antigen or antigenicepitope, may then be arranged via main-chain/side-chain linkage, viaside-chain/main-chain linkage or via side-chain/side chain linkage toeither the cell penetrating peptide (a) or to the TLR peptide agonist(c) or, if present, to an additional component like a spacer or linker,which may be, for example, positioned between the cell penetratingpeptide (a) and the TLR peptide agonist (c).

This includes the following arrangements:

-   -   (xix) component b) may be linked—optionally via a spacer or a        linker—via main-chain/side-chain linkage to component a), i.e.        the main chain of the at least one antigen or antigenic epitope        is covalently linked—optionally via a spacer or a linker—to the        side chain of the cell penetrating peptide;    -   (xx) component b) may be linked—optionally via a spacer or a        linker—via side-chain/main-chain linkage to component a), i.e.        the side chain of the at least one antigen or antigenic epitope        is covalently linked—optionally via a spacer or a linker—to the        main chain of the cell penetrating peptide;    -   (xxi) component b) may be linked—optionally via a spacer or a        linker—via side-chain/side-chain linkage to component a), i.e.        the side chain of the at least one antigen or antigenic epitope        is covalently linked—optionally via a spacer or a linker—to the        side chain of the cell penetrating peptide;    -   (xxii) component b) may be linked—optionally via a spacer or a        linker—via main-chain/side-chain linkage to component c), i.e.        the main chain of the at least one antigen or antigenic epitope        is covalently linked—optionally via a spacer or a linker—to the        side chain of the at least one TLR peptide agonist;    -   (xxiii) component b) may be linked—optionally via a spacer or a        linker—via side-chain/main-chain linkage to component c), i.e.        the side chain of the at least one antigen or antigenic epitope        is covalently linked—optionally via a spacer or a linker—to the        main chain of the at least one TLR peptide agonist;    -   (xxiv) component b) may be linked—optionally via a spacer or a        linker—via side-chain/side-chain linkage to component c), i.e.        the side chain of the at least one antigen or antigenic epitope        is covalently linked—optionally via a spacer or a linker—to the        side chain of the at least one TLR peptide agonist;    -   (xxv) component b) may be linked—optionally via a spacer or a        linker—via main-chain/side-chain linkage to a linker or a spacer        positioned between component a) and component c), i.e. the main        chain of the at least one antigen or antigenic epitope is        covalently linked—optionally via a spacer or a linker—to the        side chain of a linker or a spacer positioned between        component a) and component c);    -   (xxvi) component b) may be linked—optionally via a spacer or a        linker—via side-chain/main-chain linkage to a linker or a spacer        positioned between component a) and component c), i.e. the side        chain of the at least one antigen or antigenic epitope is        covalently linked—optionally via a spacer or a linker—to the        main chain of a linker or a spacer positioned between        component a) and component c); or    -   (xxvii) component b) may be linked—optionally via a spacer or a        linker—via side-chain/side-chain linkage to a linker or a spacer        positioned between component a) and component c), i.e. the side        chain of the at least one antigen or antigenic epitope is        covalently linked—optionally via a spacer or a linker—to the        side chain of a linker or a spacer positioned between        component a) and component c).

Alternatively, it is also conceivable that in the complex according tothe present invention all three of the components a), b), and c) arearranged via main-chain/side-chain linkage, via side-chain/main-chainlinkage or via side-chain/side chain linkage, optionally linked by anadditional component, e.g. a spacer or a linker.

Nucleic Acid Encoding the Peptides and Protein Complexes according tothe Invention

In another aspect the present invention provides a nucleic acid encodingthe complex according to the present invention, wherein the complex is apolypeptide or a protein. In particular, the present invention providespolynucleotides encoding the complex according to the present inventionas defined above.

In this context, nucleic acids preferably comprise single stranded,double stranded or partially double stranded nucleic acids, preferablyselected from genomic DNA, cDNA, RNA, siRNA, antisense DNA, antisenseRNA, ribozyme, complimentary RNA/DNA sequences with or withoutexpression elements, a mini-gene, gene fragments, regulatory elements,promoters, and combinations thereof.

Preferably, the invention relates to a nucleic acid encoding a complex,which is in particular a polypeptide or protein, said complex comprisinga cell penetrating peptide, at least one antigen or antigenic epitope,which is a polypeptide or protein, and at least one TLR peptide agonist,wherein the cell penetrating peptide, the at least one antigen orantigenic epitope, and the at least one TLR peptide agonist arecovalently linked, optionally with peptidic spacer(s) or linker(s) asdescribed herein. If more than one antigen or antigenic epitope, whichis a polypeptide or protein, is comprised by said complex, the more thanone antigens or antigenic epitopes are also covalently linked,optionally with peptidic spacer(s) or linker(s) as described herein.Similarly, if more than one TLR peptide agonist is comprised by saidcomplex, the more than one TLR peptide agonists are also covalentlylinked, optionally with peptidic spacer(s) or linker(s) as describedherein.

Particularly preferably the nucleic acid according to the presentinvention encodes a complex which is a (recombinant) fusion proteincomprising (a) a cell penetrating peptide as described above, (b) atleast one, preferably at least two, more preferably at least three, evenmore preferably at least four, particularly preferably at least five,most preferably at least six antigens or antigenic epitopes as describedabove, preferably arranged in a consecutive manner as described aboveand (c) at least one TLR agonist as described above.

Production and Purification of the Cell Penetrating Peptides andComplexes according to the Invention

According to a further aspect the present invention provides a vector,in particular a recombinant vector, comprising a nucleic acid accordingto the present invention as described above.

The term “vector”, as used in the context of the present invention,refers to a nucleic acid molecule, preferably to an artificial nucleicacid molecule, i.e. a nucleic acid molecule which does not occur innature. A vector in the context of the present invention is suitable forincorporating or harboring a desired nucleic acid sequence. Such vectorsmay be storage vectors, expression vectors, cloning vectors, transfervectors etc. A storage vector is a vector which allows the convenientstorage of a nucleic acid molecule. Thus, the vector may comprise asequence corresponding, e.g., to a desired antibody or antibody fragmentthereof according to the present invention. An expression vector may beused for production of expression products such as RNA, e.g. mRNA, orpeptides, polypeptides or proteins. For example, an expression vectormay comprise sequences needed for transcription of a sequence stretch ofthe vector, such as a promoter sequence. A cloning vector is typically avector that contains a cloning site, which may be used to incorporatenucleic acid sequences into the vector. A cloning vector may be, e.g., aplasmid vector or a bacteriophage vector. A transfer vector may be avector which is suitable for transferring nucleic acid molecules intocells or organisms, for example, viral vectors. A vector in the contextof the present invention may be, e.g., an RNA vector or a DNA vector.Preferably, a vector is a DNA molecule. For example, a vector in thesense of the present application comprises a cloning site, a selectionmarker, such as an antibiotic resistance factor, and a sequence suitablefor multiplication of the vector, such as an origin of replication.Preferably, a vector in the context of the present application is aplasmid vector. Preferably, a vector in the context of the presentapplication is an expression vector.

Cells transformed with a vector according to the present invention arealso included within the scope of the invention. Examples of such cellsinclude, but are not limited to, bacterial cells, e.g. E. coli, andeukaryotic cells, e.g., yeast cells, animal cells or plant cells. In oneembodiment the cells are mammalian, e.g., human, CHO, HEK293T, PER.C6,NS0, myeloma or hybridoma cells. Accordingly, the present invention alsorelates to a cell expressing the antibody, or the antigen bindingfragment thereof, according to the present invention; or comprising thevector according to the present invention.

In particular, a cell may be transfected with a vector according to thepresent invention, preferably with an expression vector. The term“transfection” refers to the introduction of nucleic acid molecules,such as DNA or RNA (e.g. mRNA) molecules, into cells, preferably intoeukaryotic cells. In the context of the present invention, the term“transfection” encompasses any method known to the skilled person forintroducing nucleic acid molecules into cells, preferably intoeukaryotic cells, such as into mammalian cells. Such methods encompass,for example, electroporation, lipofection, e.g. based on cationic lipidsand/or liposomes, calcium phosphate precipitation, nanoparticle basedtransfection, virus based transfection, or transfection based oncationic polymers, such as DEAE-dextran or polyethylenimine etc.Preferably, the introduction is non-viral.

Numerous expression systems can be used, including without limitationchromosomes, episomes, and derived viruses. More particularly, thevector according to the present invention, in particular the recombinantvector used, can be derived from bacterial plasmids, transposons, yeastepisomes, insertion elements, yeast chromosome elements, viruses such asbaculovirus, papilloma viruses such as SV40, vaccinia viruses,adenoviruses, fox pox viruses, pseudorabies viruses, retroviruses.

For example, such vectors, in particular recombinant vectors, canequally be cosmid or phagemid derivatives. The nucleotide sequence, inparticular the nucleic acid according to the present invention, may beinserted in the recombinant expression vector by methods well known to aperson skilled in the art such as, for example, those described inMOLECULAR CLONING: A LABORATORY MANUAL, Sambrook et al., 4th Ed., ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 2001.

The vector, in particular the recombinant vector, may also includenucleotide sequences that control the regulation of the expression, inparticular of the nucleic acid according to the present invention, aswell as nucleotide sequences permitting the expression, thetranscription, and the translation, in particular of the nucleic acidaccording to the present invention. Typically, these sequences areselected according to the host cells used.

Thus, for example, an appropriate secretion signal can be integrated inthe vector according to the present invention, in particular in arecombinant vector, so that the polypeptide or protein encoded by thenucleic acid according to the present invention, will be directed, forexample towards the lumen of the endoplasmic reticulum, towards theperiplasmic space, on the membrane or towards the extracellularenvironment. The choice of an appropriate secretion signal mayfacilitate subsequent protein purification.

In yet another aspect the present invention provides a host cellcomprising a vector, in particular a recombinant vector, according tothe present invention.

The introduction of the vector, in particular the recombinant vector,according to the present invention into a host cell can be carried outaccording to methods that are well known to a person skilled in the art,such as those described in BASIC METHODS IN MOLECULAR BIOLOGY, Davis etal., 2nd ed., McGraw-Hill Professional Publishing, 1995, and MOLECULARCLONING: A LABORATORY MANUAL, supra, including for example transfectionas described above, e.g. by calcium phosphate, by DEAE dextran, or bycationic lipids; microinjection, electroporation, transduction orinfection.

The host cell can be, for example, bacterial cells such as E. coli,cells of fungi such as yeast cells and cells of Aspergillus,Streptomyces, insect cells, and/or any cell line, e.g. Chinese HamsterOvary cells (CHO), C127 mouse cell line, BHK cell line of Syrian hamstercells, Human Embryonic Kidney 293 (HEK 293) cells. Preferably, the hostcell according to the present invention is mammalian, e.g., human, CHO,HEK293T, PER.C6, NS0, myeloma or hybridoma cells. Dendritic cells anddendritic cell lines are particularly preferred as a host cell.Typically, the choice of a culture medium depends in particular on thechoice of the cell type and/or the cell line, whereby the skilled personis aware of suitable culture media, which are appropriate for a selectedcell type and/or cell line.

The host cells can be used, for example, to express a polypeptide orprotein, in particular the complex according to the present invention,on the basis of the vector and/or the nucleic acid according to thepresent invention. After purification by standard methods, the expressedpolypeptide or protein, in particular the complex according to thepresent invention, can be used in a method as described hereinafter.

Accordingly, the present invention also provides a method for preparingthe complex according to the present invention, in particular whereinthe complex is a polypeptide or protein. Said method comprises the stepsof:

-   -   (i) cultivating a host cell as described above in a culture        medium; and    -   (ii) separating the complex according to the present invention        from the culture medium or separating the complex according to        the present invention from the host cell lysate after host cell        lysis.

Thus, the complex obtained by such a method according to the presentinvention is preferably a complex according to the present invention asdescribed herein.

For protein extraction commercially available kits and/or reagents maybe used, for example BugBuster™ from Novagen.

Preferably, the method for preparing the complex according to thepresent invention as described above further comprises the followingstep:

-   -   (iii) solubilization of the complex according to the present        invention, e.g. by resuspension in solutions containing urea or        guanidine hydrochloride (GuHCl),

wherein step (iii) follows step (ii) as described above.

Moreover, it is preferred that the method for preparing the complexaccording to the present invention as described above further comprisesthe following step:

-   -   (iv) purification of the complex according to the present        invention, preferably by one-step affinity chromatography,

wherein step (iv) follows step (ii), or, if present, step (iii) asdescribed above.

In addition, the complex according to the present invention may also beprepared by synthetic chemistry methods, for example by solid-phasepeptide synthesis.

Purification of those peptides or proteins may be carried out by meansof any technique known in the art for protein/peptide purification.Exemplary techniques include ion-exchange chromatography, hydrophobicinteraction chromatography, and immunoaffinity methods.

Thus, the present invention also provides a method for preparing thecomplex according to the present invention comprising the steps of:

-   -   (i) chemically synthesizing said complex; and    -   (ii) purifying said complex.

Preferably, in the method for preparing a complex according to thepresent invention, the complex chemically synthesized in step (i) andpurified in step (ii) comprises an amino acid sequences as describedherein for a cell penetrating peptide, an amino acid sequence asdescribed herein for a TLR peptide agonist, and, optionally if the atleast one antigen and/or antigenic epitope is a peptide or a protein, anamino acid sequence as described herein for an antigen or antigenicepitope.

Alternatively, the present invention also provides a method forpreparing the complex according to the present invention, wherein

-   -   (i) the cell penetrating peptide, the at least one antigen or        antigenic fragment and/or the at least one TLR peptide agonist        are synthesized separately;    -   (ii) optionally, the cell penetrating peptide, the at least one        antigen or antigenic fragment and/or the at least one TLR        peptide agonist are purified; and    -   (iii) the cell penetrating peptide, the at least one antigen or        antigenic fragment and/or the at least one TLR peptide agonist        are covalently linked as described above, optionally by a spacer        or linker or by a cross-linking agent as described above.

Cells Loaded with the Complexes according to the Invention

In yet another aspect the present invention relates to a cell loadedwith the complex according to the invention. For example, the cellsloaded with the complex according to the invention are cells from asubject to be treated, in particular isolated cells from a subject to betreated, i.e. cells isolated from a subject to be treated.

As used in the context of the present invention, the term “subject”refers in particular to mammals. For example, mammals contemplated bythe present invention include human, primates, domesticated animals suchas cattle, sheep, pigs, horses, laboratory rodents and the like. Morepreferably, the term “subject” refers to a human subject.

As used in the context of the present invention, “treatment” and“treating” and the like generally mean obtaining a desiredpharmacological and physiological effect. The effect may be prophylacticin terms of preventing or partially preventing a disease, a symptom or acondition thereof and/or may be therapeutic in terms of a partial orcomplete cure of a disease, a condition, a symptom or an adverse effectattributed to the disease. The term “treatment” as used herein coversany treatment of a disease in a mammal, in particular in a human, andincludes: (a) preventing the disease from occurring in a subject who maybe predisposed to the disease but the outbreak of the disease has notyet occurred and/or the disease has not yet been diagnosed in thissubject, for example a preventive early asymptomatic intervention; (b)inhibiting the disease, i.e., arresting or slowing down its development;or (c) relieving the disease, i.e., causing an at least partialregression of the disease and/or of at least one of its symptoms orconditions such as improvement or remediation of damage. In particular,the methods, uses, formulations and compositions according to theinvention are useful in the treatment of cancers or infectious diseasesand/or in the prevention of evolution of cancers into an advanced ormetastatic stage in subjects with early stage cancer, thereby improvingthe staging of the cancer. When applied to cancers, prevention of adisease or disorder includes the prevention of the appearance ordevelopment of a cancer in an individual identified as at risk ofdeveloping said cancer, for instance due to past occurrence of saidcancer in the circle of the individual's relatives, and prevention ofinfection with tumor promoting pathogens such as, for example,Epstein-Barr virus (EBV), Human papillomavirus (HPV), Hepatitis B virus(HBV), Hepatitis C virus (HCV), Human Herpes virus 8 (HHV8), humanT-cell leukemia virus type 1 (HTLV-1), Merkel cell polyomavirus (MCV)and Helicobacter pylori. Also covered by the terms“prevention/treatment” of a cancer is the stabilization or delay of analready diagnosed cancer in an individual. By “stabilization”, it ismeant the prevention of evolution of cancer into advanced or metastaticstage in subjects with early stage cancer.

Preferably, the cell loaded with the complex according to the inventionis an antigen-presenting cell (APC). Preferably, the antigen presentingcell is selected from the group consisting of a dendritic cell (DC), amacrophage and a B-cell. Dendritic cells, in particular dendritic cells(conventional and/or plasmacytoid) isolated from a subject to betreated, are more preferred.

Methods to isolate antigen-presenting cells, in particular dendriticcells, from a subject are known to the skilled person. They includeharvesting monocytes or hematopoietic stem cells from bone marrow, cordblood, or peripheral blood. They also include the use of embryonic stem(ES) cells and induced pluripotent stem cells (iPS). Antigen presentingcells, in particular dendritic cells or their precursors, can beenriched by methods including elutriation and magnetic bead basedseparation, which may involve enrichment for CD14⁺ precursor cells.

Methods to load the complex according to the present invention into thecells, preferably into the above-mentioned antigen presenting cells,more preferably into dendritic cells, and further to prepare such cellsbefore administration to a subject are known to one skilled in the art.For example, preparation of dendritic cells can include their culture ordifferentiation using cytokines that may include for example GM-CSF andIL-4. Dendritic cell lines may also be employed. Loading of the complexof the invention into the cells, preferably into APC, more preferablyinto the dendritic cells, can involve co-incubation of the complex ofthe invention with the cells in culture, making use of the intrinsicproperties of the cell penetrating peptide comprised by the complexaccording to the invention (i.e. its internalization ability). Furtherculture of the cells, e.g. the dendritic cells, thus loaded to induceefficient maturation can include addition of cytokines including IL-1β,IL-6, TNFα, PGE2, IFNα, and adjuvants which may include poly-IC,poly-ICLC (i.e. a synthetic complex of carboxymethylcellulose,polyinosinic-polycytidylic acid, and poly-L-lysine double-stranded RNA),and further TLR agonists and NLR (nucleotide-binding oligomerizationdomain-like receptors) agonists.

According to a further aspect the present invention also concernsimaging cells used for cell therapy, such as stem cells, dendriticcells, T cells or natural killer cells, wherein the cells are loadedwith the complex according to the invention, which may further comprisean imaging agent.

The present invention also provides a method for preparing cells, inparticular antigen presenting cells, loaded with the complex accordingto the present invention as described above, said method comprising thesteps of:

-   -   (i) transducing or transfecting said cells with the complex of        the invention;    -   (ii) cultivating said cells in a culture medium; and    -   (iii) separating said cells from the culture medium.

Preferably, the cells are loaded with a complex according to the presentinvention, wherein the complex is a polypeptide or a protein.

Preferably, the cells loaded with a complex(es) according to the presentinvention present the at least one antigen or antigenic epitopecomprised by said complex at the cell surface in an MHC class I contextand/or in an MHC class II context.

Compositions and Kits according to the Present Invention

According to another aspect, the invention provides a compositioncomprising at least one component selected from:

-   -   (i) a complex according to the present invention as described        above,    -   (ii) a nucleic acid according to the present invention as        described above,    -   (iii) a vector according to the present invention as described        above,    -   (iv) a host cell according to the present invention as described        above, and    -   (v) a cell loaded with a complex according to the present        invention as described above.

Preferably, the composition according to the present invention comprisesthe complex according to the present invention as described above.

The composition according to the present invention may also comprisesmore than one of the above components (i) to (v). For example, thecomposition according to the present invention may comprise at least twodifferent complexes under (i), at least two different nucleic acidsunder (ii), at least two different vectors under (iii), at least twodifferent host cells under (iv), and/or at least two different cellsunder (v); e.g., the composition of the invention may comprise at leasttwo different complexes (i) and/or at least two different nucleic acids(ii).

For example, the different complexes (i) comprised by the composition asdescribed above may differ in either component a), i.e. in the cellpenetrating peptides, in component b), i.e. in the antigens or antigenicepitopes or in the subsets of more than one antigen or antigenicepitope, or in component c), i.e. in the TLR peptide agonist or in thesubset of more than one TLR peptide agonist; or the different complexes(i) comprised by the composition as described above may differ in twoout of the three components a), b), and c); or the different complexes(i) comprised by the composition as described above may differ in allthree components a), b), and c) of the complex. Accordingly, thedifferent nucleic acids (ii) comprised by the composition as describedabove may differ in that they encode such different complexes; thedifferent vectors (iii) comprised by the composition as described abovemay differ in that they comprise such different nucleic acids; thedifferent host cells (iv) comprised by the composition as describedabove may differ in that they comprise such different vectors; and thedifferent cells loaded with a complex (v) comprised by the compositionas described above may differ in that they are loaded with suchdifferent complexes.

The present invention also provides a complex or cells loaded with saidcomplex, as described herewith, for use as a medicament, in particularas a vaccine. In particular, the composition according to the presentinvention is preferably a vaccine.

Thus, the present invention also provides a vaccine comprising at leastone component selected from:

-   -   (i) a complex according to the present invention as described        above,    -   (ii) a nucleic acid according to the present invention as        described above,    -   (iii) a vector according to the present invention as described        above,    -   (iv) a host cell according to the present invention as described        above, and    -   (v) a cell loaded with a complex according to the present        invention as described above.

Preferably, the vaccine according to the present invention comprises thecomplex according to the present invention as described above.

Thereby, the above details described for the composition according tothe present invention regarding more than one of the components (i) to(v), also apply for the vaccine according to the present invention.

As used in the context of the present invention, the term “vaccine”refers to a biological preparation that provides innate and/or adaptiveimmunity, typically to a particular disease, preferably cancer. Thus, avaccine supports in particular an innate and/or an adaptive immuneresponse of the immune system of a subject to be treated. For example,the antigen or antigenic epitope of the complex according to the presentinvention typically leads to or supports an adaptive immune response inthe patient to be treated, and the TLR peptide agonist of the complexaccording to the present invention may lead to or support an innateimmune response.

The inventive composition, in particular the inventive vaccine, may alsocomprise a pharmaceutically acceptable carrier, adjuvant, and/or vehicleas defined below for the inventive pharmaceutical composition. In thespecific context of the inventive composition, in particular of theinventive vaccine, the choice of a pharmaceutically acceptable carrieris determined in principle by the manner in which the inventivecomposition, in particular the inventive vaccine, is administered. Theinventive composition, in particular the inventive vaccine, can beadministered, for example, systemically or locally. Routes for systemicadministration in general include, for example, transdermal, oral,parenteral routes, including subcutaneous, intravenous, intramuscular,intraarterial, intradermal and intraperitoneal injections and/orintranasal administration routes. Routes for local administration ingeneral include, for example, topical administration routes but alsointradermal, transdermal, subcutaneous, or intramuscular injections orintralesional, intracranial, intrapulmonal, intracardial, intranodal andsublingual injections. More preferably, inventive composition, inparticular the vaccines, may be administered by an intradermal,subcutaneous, intranodal or intramuscular route. Even more preferably,the inventive composition, in particular the vaccine, may beadministered by subcutaneous, intranodal or intramuscular route.Particularly preferably, the inventive composition, in particular thevaccines, may be administered by subcutaneous or intranodal route. Mostpreferably, the inventive composition, in particular the vaccines may beadministered by subcutaneous route. Inventive composition, in particularthe inventive vaccines, are therefore preferably formulated in liquid(or sometimes in solid) form.

The suitable amount of the inventive composition, in particular theinventive vaccine, to be administered can be determined by routineexperiments with animal models. Such models include, without implyingany limitation, rabbit, sheep, mouse, rat, dog and non-human primatemodels. Preferred unit dose forms for injection include sterilesolutions of water, physiological saline or mixtures thereof. The pH ofsuch solutions should be adjusted to about 7.4. Suitable carriers forinjection include hydrogels, devices for controlled or delayed release,polylactic acid and collagen matrices. Suitable pharmaceuticallyacceptable carriers for topical application include those which aresuitable for use in lotions, creams, gels and the like. If the inventivecomposition, in particular the inventive vaccine, is to be administeredorally, tablets, capsules and the like are the preferred unit dose form.The pharmaceutically acceptable carriers for the preparation of unitdose forms which can be used for oral administration are well known inthe prior art. The choice thereof will depend on secondaryconsiderations such as taste, costs and storability, which are notcritical for the purposes of the present invention, and can be madewithout difficulty by a person skilled in the art.

The inventive composition, in particular the inventive vaccine, canadditionally contain one or more auxiliary substances in order tofurther increase its immunogenicity. A synergistic action of theinventive complex as defined above and of an auxiliary substance, whichmay be optionally contained in the inventive vaccine as described above,is preferably achieved thereby. Depending on the various types ofauxiliary substances, various mechanisms can come into consideration inthis respect. For example, compounds that permit the maturation ofdendritic cells (DCs), for example lipopolysaccharides, TNF-alpha orCD40 ligand, form a first class of suitable auxiliary substances. Ingeneral, it is possible to use as auxiliary substance any agent thatinfluences the immune system in the manner of a “danger signal” (LPS,GP96, etc.) or cytokines, such as GM-CSF, which allow an immune responseproduced by the immune-stimulating adjuvant according to the inventionto be enhanced and/or influenced in a targeted manner. Particularlypreferred auxiliary substances are cytokines, such as monokines,lymphokines, interleukins or chemokines, that further promote the innateimmune response, such as IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8,IL-9, IL-10, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19,IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29,IL-30, IL-31, IL-32, IL-33, IFN-alpha, IFN-beta, IFN-gamma, GM-CSF,G-CSF, M-CSF, LT-beta or TNF-alpha, growth factors, such as hGH.

Further additives which may be included in the inventive vaccine areemulsifiers, such as, for example, Tween®; wetting agents, such as, forexample, sodium lauryl sulfate; colouring agents; taste-impartingagents, pharmaceutical carriers; tablet-forming agents; stabilizers;antioxidants; preservatives.

The inventive composition, in particular the inventive vaccine, can alsoadditionally contain any further compound, which is known to beimmune-stimulating due to its binding affinity (as ligands) to humanToll-like receptors TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8,TLR9, TLR10, or due to its binding affinity (as ligands) to murineToll-like receptors TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8,TLR9, TLR10, TLR11, TLR12 or TLR13.

Another class of compounds, which may be added to an inventivecomposition, in particular to an inventive vaccine, in this context, maybe CpG nucleic acids, in particular CpG-RNA or CpG-DNA. A CpG-RNA orCpG-DNA can be a single-stranded CpG-DNA (ss CpG-DNA), a double-strandedCpG-DNA (dsDNA), a single-stranded CpG-RNA (ss CpG-RNA) or adouble-stranded CpG-RNA (ds CpG-RNA). The CpG nucleic acid is preferablyin the form of CpG-RNA, more preferably in the form of single-strandedCpG-RNA (ss CpG-RNA). The CpG nucleic acid preferably contains at leastone or more (mitogenic) cytosine/guanine dinucleotide sequence(s) (CpGmotif(s)). According to a first preferred alternative, at least one CpGmotif contained in these sequences, in particular the C (cytosine) andthe G (guanine) of the CpG motif, is unmethylated. All further cytosinesor guanines optionally contained in these sequences can be eithermethylated or unmethylated. According to a further preferredalternative, however, the C (cytosine) and the G (guanine) of the CpGmotif can also be present in methylated form.

Moreover, the present invention also provides the complex as describedabove or cells loaded with said complex or a composition or a vaccine asdescribed above for use in the prevention and/or treatment of diseasesor disorders including for example cancers, hematological disorders,infectious diseases, autoimmunity disorders and transplant rejections,whereby cancer is preferred.

In addition, the present invention also provides the complex asdescribed above or cells loaded with said complex or a composition asdescribed above for use as an imaging or diagnostic composition.

The present invention also provides a pharmaceutical composition, inparticular a vaccine composition as described above, and a method fortreating a subject, preferably a mammalian subject, and most preferablya human subject, who is suffering from a disease or disorder, inparticular from a disorder that can be treated by immunotherapy such ascancers, infectious diseases, autoimmunity disorders and transplantrejections.

In particular, the present invention provides a pharmaceuticalcomposition comprising at least one complex according to the presentinvention or at least one cell loaded with a complex according to thepresent invention, and optionally a pharmaceutically acceptable carrierand/or vehicle, or any excipient, buffer, stabilizer or other materialswell known to those skilled in the art, in particular the pharmaceuticalcomposition comprising at least one complex according to the presentinvention or at least one cell loaded with a complex according to thepresent invention and a pharmaceutically acceptable carrier.

As a further ingredient, the inventive pharmaceutical composition may inparticular comprise a pharmaceutically acceptable carrier and/orvehicle. In the context of the present invention, a pharmaceuticallyacceptable carrier typically includes the liquid or non-liquid basis ofthe inventive pharmaceutical composition. If the inventivepharmaceutical composition is provided in liquid form, the carrier willtypically be pyrogen-free water; isotonic saline or buffered (aqueous)solutions, e.g phosphate, citrate etc. buffered solutions. Particularlyfor injection of the inventive inventive pharmaceutical composition,water or preferably a buffer, more preferably an aqueous buffer, may beused, containing a sodium salt, preferably at least 30 mM of a sodiumsalt, a calcium salt, preferably at least 0.05 mM of a calcium salt, andoptionally a potassium salt, preferably at least 1 mM of a potassiumsalt. According to a preferred embodiment, the sodium, calcium and,optionally, potassium salts may occur in the form of their halogenides,e.g. chlorides, iodides, or bromides, in the form of their hydroxides,carbonates, hydrogen carbonates, or sulfates, etc. Without being limitedthereto, examples of sodium salts include e.g. NaCl, NaI, NaBr, Na₂CO₃,NaHCO₃, Na₂SO₄, examples of the optional potassium salts include e.g.KCl, KI, KBr, K₂CO₃, KHCO₃, K₂SO₄, and examples of calcium salts includee.g. CaCl₂, CaI₂, CaBr₂, CaCO₃, CaSO₄, Ca(OH)₂. Furthermore, organicanions of the aforementioned cations may be contained in the buffer.According to a more preferred embodiment, the buffer suitable forinjection purposes as defined above, may contain salts selected fromsodium chloride (NaCl), calcium chloride (CaCl₂) and optionallypotassium chloride (KCl), wherein further anions may be presentadditional to the chlorides. CaCl₂ can also be replaced by another saltlike KCl. Typically, the salts in the injection buffer are present in aconcentration of at least 30 mM sodium chloride (NaCl), at least 1 mMpotassium chloride (KCl) and at least 0.05 mM calcium chloride (CaCl₂).The injection buffer may be hypertonic, isotonic or hypotonic withreference to the specific reference medium, i.e. the buffer may have ahigher, identical or lower salt content with reference to the specificreference medium, wherein preferably such concentrations of the aforementioned salts may be used, which do not lead to damage of cells due toosmosis or other concentration effects. Reference media are e.g. liquidsoccurring in “in vivd” methods, such as blood, lymph, cytosolic liquids,or other body liquids, or e.g. liquids, which may be used as referencemedia in “in vitro” methods, such as common buffers or liquids. Suchcommon buffers or liquids are known to a skilled person. Saline (0.9%NaCl) and Ringer-Lactate solution are particularly preferred as a liquidbasis.

However, one or more compatible solid or liquid fillers or diluents orencapsulating compounds may be used as well for the inventivepharmaceutical composition, which are suitable for administration to asubject to be treated. The term “compatible” as used herein means thatthese constituents of the inventive pharmaceutical composition arecapable of being mixed with the complex according to the presentinvention as defined above in such a manner that no interaction occurswhich would substantially reduce the pharmaceutical effectiveness of theinventive pharmaceutical composition under typical use conditions.Pharmaceutically acceptable carriers, fillers and diluents must, ofcourse, have sufficiently high purity and sufficiently low toxicity tomake them suitable for administration to a subject to be treated. Someexamples of compounds which can be used as pharmaceutically acceptablecarriers, fillers or constituents thereof are sugars, such as, forexample, lactose, glucose and sucrose; starches, such as, for example,corn starch or potato starch; cellulose and its derivatives, such as,for example, sodium carboxymethylcellulose, ethylcellulose, celluloseacetate; powdered tragacanth; malt; gelatin; tallow; solid glidants,such as, for example, stearic acid, magnesium stearate; calcium sulfate;vegetable oils, such as, for example, groundnut oil, cottonseed oil,sesame oil, olive oil, corn oil and oil from theobroma; polyols, suchas, for example, polypropylene glycol, glycerol, sorbitol, mannitol andpolyethylene glycol; alginic acid.

The inventive pharmaceutical composition may be administered orally,parenterally, by inhalation spray, topically, rectally, nasally,buccally, vaginally or via an implanted reservoir. The term parenteralas used herein includes subcutaneous, intravenous, intramuscular,intra-articular, intra-synovial, intrasternal, intrathecal,intrahepatic, intralesional, intracranial, transdermal, intradermal,intrapulmonal, intraperitoneal, intracardial, intraarterial, intranodaland sublingual injection or infusion techniques. Preferably, theinventive pharmaceutical composition may be administered intradermally,intramuscularly, intranodally or subcutaneously. More preferably theinventive pharmaceutical composition may be administeredintramuscularly, intranodally or subcutaneously. Even more preferablythe inventive pharmaceutical composition may be administeredintranodally or subcutaneously. Most preferably, the inventivepharmaceutical composition may be administered subcutaneously.

Preferably, the inventive pharmaceutical composition may be administeredby parenteral injection, more preferably by subcutaneous, intravenous,intramuscular, intra-articular, intra-synovial, intrasternal,intrathecal, intrahepatic, intralesional, intracranial, transdermal,intradermal, intrapulmonal, intraperitoneal, intracardial,intraarterial, intranodal and sublingual injection or via infusiontechniques. Sterile injectable forms of the inventive pharmaceuticalcompositions may be aqueous or oleaginous suspension. These suspensionsmay be formulated according to techniques known in the art usingsuitable dispersing or wetting agents and suspending agents. The sterileinjectable preparation may also be a sterile injectable solution orsuspension in a non-toxic parenterally-acceptable diluent or solvent,for example as a solution in 1.3-butanediol. Among the acceptablevehicles and solvents that may be employed are water, Ringer's solutionand isotonic sodium chloride solution. In addition, sterile, fixed oilsare conventionally employed as a solvent or suspending medium. For thispurpose, any bland fixed oil may be employed including synthetic mono-or di-glycerides. Fatty acids, such as oleic acid and its glyceridederivatives are useful in the preparation of injectables, as are naturalpharmaceutically-acceptable oils, such as olive oil or castor oil,especially in their polyoxyethylated versions. These oil solutions orsuspensions may also contain a long-chain alcohol diluent or dispersant,such as carboxymethyl cellulose or similar dispersing agents that arecommonly used in the formulation of pharmaceutically acceptable dosageforms including emulsions and suspensions. Other commonly usedsurfactants, such as Tweens, Spans and other emulsifying agents orbioavailability enhancers which are commonly used in the manufacture ofpharmaceutically acceptable solid, liquid, or other dosage forms mayalso be used for the purposes of formulation of the inventivepharmaceutical composition.

For intravenous, cutaneous or subcutaneous injection, or injection atthe site of affliction, the active ingredient will preferably be in theform of a parenterally acceptable aqueous solution which is pyrogen-freeand has suitable pH, isotonicity and stability. Those of relevant skillin the art are well able to prepare suitable solutions using, forexample, isotonic vehicles such as Sodium Chloride Injection, Ringer'sInjection, Lactated Ringer's Injection. Preservatives, stabilizers,buffers, antioxidants and/or other additives may be included, asrequired. Whether it is a polypeptide, peptide, or nucleic acidmolecule, other pharmaceutically useful compound according to thepresent invention that is to be given to an individual, administrationis preferably in a “prophylactically effective amount” or a“therapeutically effective amount” (as the case may be), this beingsufficient to show benefit to the individual. The actual amountadministered, and rate and time-course of administration, will depend onthe nature and severity of what is being treated.

The inventive pharmaceutical composition as defined above may also beadministered orally in any orally acceptable dosage form including, butnot limited to, capsules, tablets, aqueous suspensions or solutions. Inthe case of tablets for oral use, carriers commonly used include lactoseand corn starch. Lubricating agents, such as magnesium stearate, arealso typically added. For oral administration in a capsule form, usefuldiluents include lactose and dried cornstarch. When aqueous suspensionsare required for oral use, the active ingredient, i.e. the inventivetransporter cargo conjugate molecule as defined above, is combined withemulsifying and suspending agents. If desired, certain sweetening,flavoring or coloring agents may also be added.

The inventive pharmaceutical composition may also be administeredtopically, especially when the target of treatment includes areas ororgans readily accessible by topical application, e.g. includingdiseases of the skin or of any other accessible epithelial tissue.Suitable topical formulations are readily prepared for each of theseareas or organs. For topical applications, the inventive pharmaceuticalcomposition may be formulated in a suitable ointment, containing theinventive immunostimulatory composition, particularly its components asdefined above, suspended or dissolved in one or more carriers. Carriersfor topical administration include, but are not limited to, mineral oil,liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene,polyoxypropylene compound, emulsifying wax and water. Alternatively, theinventive pharmaceutical composition can be formulated in a suitablelotion or cream. In the context of the present invention, suitablecarriers include, but are not limited to, mineral oil, sorbitanmonostearate, polysorbate 60, cetyl esters wax, cetearyl alcohol,2-octyldodecanol, benzyl alcohol and water.

In this context, prescription of treatment, e.g. decisions on dosageetc. when using the above pharmaceutical composition is typically withinthe responsibility of general practitioners and other medical doctors,and typically takes account of the disorder to be treated, the conditionof the individual patient, the site of delivery, the method ofadministration and other factors known to practitioners. Examples of thetechniques and protocols mentioned above can be found in REMINGTON'SPHARMACEUTICAL SCIENCES, 16th edition, Osol, A. (ed), 1980.

Accordingly, the inventive pharmaceutical composition typicallycomprises a “safe and effective amount” of the components of theinventive pharmaceutical composition, in particular of the complexaccording to the present invention as defined above and/or cells loadedwith said complex. As used herein, a “safe and effective amount” meansan amount of the complex according to the present invention that issufficient to significantly induce a positive modification of a diseaseor disorder, i.e. an amount of the complex according to the presentinvention or cells loaded with said complex, that elicits the biologicalor medicinal response in a tissue, system, animal or human that is beingsought. An effective amount may be a “therapeutically effective amount”for the alleviation of the symptoms of the disease or condition beingtreated and/or a “prophylactically effective amount” for prophylaxis ofthe symptoms of the disease or condition being prevented. The term alsoincludes the amount of active complex sufficient to reduce theprogression of the disease, notably to reduce or inhibit the tumorgrowth or infection and thereby elicit the response being sought, inparticular such response could be an immune response directed againstthe antigens or antigenic epitopes comprised in by the complex (i.e. an“inhibition effective amount”). At the same time, however, a “safe andeffective amount” is small enough to avoid serious side-effects, that isto say to permit a sensible relationship between advantage and risk. Thedetermination of these limits typically lies within the scope ofsensible medical judgment. A “safe and effective amount” of thecomponents of the inventive pharmaceutical composition, particularly ofthe complex according to the present invention as defined above, willfurthermore vary in connection with the particular condition to betreated and also with the age and physical condition of the patient tobe treated, the body weight, general health, sex, diet, time ofadministration, rate of excretion, drug combination, the activity of thespecific components a), b), and c) of the complex according to thepresent invention as defined above, the severity of the condition, theduration of the treatment, the nature of the accompanying therapy, ofthe particular pharmaceutically acceptable carrier used, and similarfactors, within the knowledge and experience of the accompanying doctor.The inventive pharmaceutical composition may be used for human and alsofor veterinary medical purposes, preferably for human medical purposes,as a pharmaceutical composition in general or as a vaccine.

Pharmaceutical compositions, in particular vaccine compositions, orformulations according to the invention may be administered as apharmaceutical formulation which can contain a complex according to thepresent invention in any form described herein.

The terms “pharmaceutical formulation” and “pharmaceutical composition”as used in the context of the present invention refer in particular topreparations which are in such a form as to permit biological activityof the active ingredient(s) to be unequivocally effective and whichcontain no additional component which would be toxic to subjects towhich the said formulation would be administered.

In the context of the present invention, an “efficacy” of a treatmentcan be measured based on changes in the course of a disease in responseto a use or a method according to the present invention. For example,the efficacy of a treatment of cancer can be measured by a reduction oftumor volume, and/or an increase of progression free survival time,and/or a decreased risk of relapse post-resection for primary cancer.More specifically for cancer treated by immunotherapy, assessment ofefficacy can be by the spectrum of clinical patterns of antitumorresponse for immunotherapeutic agents through novel immune-relatedresponse criteria (irRC), which are adapted from Response EvaluationCriteria in Solid Tumors (RECIST) and World Health Organization (WHO)criteria (J. Natl. Cancer Inst. 2010, 102(18): 1388-1397). The efficacyof prevention of infectious disease is ultimately assessed byepidemiological studies in human populations, which often correlateswith titres of neutralizing antibodies in sera, and induction ofmultifunctional pathogen specific T cell responses. Preclinicalassessment can include resistance to infection after challenge withinfectious pathogen. Treatment of an infectious disease can be measuredby inhibition of the pathogen's growth or elimination of the pathogen(and, thus, absence of detection of the pathogen), correlating withpathogen specific antibodies and/or T cell immune responses.

Pharmaceutical compositions, in particular vaccine compositions, orformulations according to the invention may also be administered as apharmaceutical formulation which can contain antigen presenting cellsloaded with a complex according to the invention in any form describedherein.

The vaccine and/or the composition according to the present inventionmay also be formulated as pharmaceutical compositions and unit dosagesthereof, in particular together with a conventionally employed adjuvant,immunomodulatory material, carrier, diluent or excipient as describedabove and below, and in such form may be employed as solids, such astablets or filled capsules, or liquids such as solutions, suspensions,emulsions, elixirs, or capsules filled with the same, all for oral use,or in the form of sterile injectable solutions for parenteral (includingsubcutaneous and intradermal) use by injection or continuous infusion.

In the context of the present invention, in particular in the context ofa pharmaceutical composition and vaccines according to the presentinvention, injectable compositions are typically based upon injectablesterile saline or phosphate-buffered saline or other injectable carriersknown in the art. Such pharmaceutical compositions and unit dosage formsthereof may comprise ingredients in conventional proportions, with orwithout additional active compounds or principles, and such unit dosageforms may contain any suitable effective amount of the active ingredientcommensurate with the intended daily dosage range to be employed.

Examples of suitable adjuvants and/or immunomodulatory materials in thecontext of the present invention include MPL® (Corixa), aluminum-basedminerals including aluminum compounds (generically called Alum), ASO1-4,MF59, CalciumPhosphate, Liposomes, Iscom, polyinosinic:polycytidylicacid (polylC), including its stabilized form poly-ICLC (Hiltonol), CpGoligodeoxynucleotides, Granulocyte-macrophage colony-stimulating factor(GM-CSF), lipopolysaccharide (LPS), Montanide, polylactide co-glycolide(PLG), Flagellin, Soap Bark tree saponins (QS21), amino alkylglucosamide compounds (e.g. RC529), two component antibacterial peptideswith synthetic oligodeoxynucleotides (e.g. IC31), Imiquimod, Resiquimod,Immunostimulatory sequences (ISS), monophosphoryl lipid A (MPLA),Fibroblast-stimulating lipopeptide (FSL1), and anti-CD40 antibodies.

Compositions, in particular pharmaceutical compositions and vaccines,according to the present invention may be liquid formulations including,but not limited to, aqueous or oily suspensions, solutions, emulsions,syrups, and elixirs. The compositions may also be formulated as a dryproduct for reconstitution with water or other suitable vehicle beforeuse. Such liquid preparations may contain additives including, but notlimited to, suspending agents, emulsifying agents, non-aqueous vehiclesand preservatives. Suspending agents include, but are not limited to,sorbitol syrup, methyl cellulose, glucose/sugar syrup, gelatin,hydroxyethyl cellulose, carboxymethyl cellulose, aluminum stearate gel,and hydrogenated edible fats. Emulsifying agents include, but are notlimited to, lecithin, sorbitan monooleate, and acacia. Preservativesinclude, but are not limited to, methyl or propyl p-hydroxybenzoate andsorbic acid. Dispersing or wetting agents include but are not limited topoly(ethylene glycol), glycerol, bovine serum albumin, Tween®, Span®.

Compositions, in particular pharmaceutical compositions and vaccines,according to the present invention may also be formulated as a depotpreparation, which may be administered by implantation or byintramuscular injection.

Compositions, in particular pharmaceutical compositions and vaccines,according to the present invention may also be solid compositions, whichmay be in the form of tablets or lozenges formulated in a conventionalmanner. For example, tablets and capsules for oral administration maycontain conventional excipients including, but not limited to, bindingagents, fillers, lubricants, disintegrants and wetting agents. Bindingagents include, but are not limited to, syrup, accacia, gelatin,sorbitol, tragacanth, mucilage of starch and polyvinylpyrrolidone.Fillers include, but are not limited to, lactose, sugar,microcrystalline cellulose, maizestarch, calcium phosphate, andsorbitol. Lubricants include, but are not limited to, magnesiumstearate, stearic acid, talc, polyethylene glycol, and silica.Disintegrants include, but are not limited to, potato starch and sodiumstarch glycollate.

Wetting agents include, but are not limited to, sodium lauryl sulfate.Tablets may be coated according to methods well known in the art.

Compositions, in particular pharmaceutical compositions and vaccines,according to the present invention may also be administered in sustainedrelease forms or from sustained release drug delivery systems.

Moreover, the compositions, in particular pharmaceutical compositionsand vaccines, according to the present invention may be adapted fordelivery by repeated administration.

Further materials as well as formulation processing techniques and thelike, which are useful in the context of compositions, in particularpharmaceutical compositions and vaccines, according to the presentinvention or in the context of their preparation are set out in “Part 5of Remington's “The Science and Practice of Pharmacy”, 22^(nd) Edition,2012, University of the Sciences in Philadelphia, Lippincott Williams &Wilkins”.

According to still another aspect, the present invention provides amethod of preparing a pharmaceutical composition according to thepresent invention comprising the step of mixing complex according to thepresent invention or cells, in particular antigen-presenting cells,loaded with a complex according to the present invention, and apharmaceutically acceptable carrier.

Accordingly, the complex according to the present invention and the cellloaded with a complex according to the present invention may be used(for the preparation of a pharmaceutical composition and/or thepreparation of a vaccine) for the prevention, treatment and/oramelioration of any of the diseases and disorders as defined herein, inparticular those that can be treated or prevented by immunotherapy suchas cancers and infectious diseases.

In another aspect, the invention provides imaging or diagnosiscompositions (“diagnostic compositions”). A still other aspect concernsmethods for delivering an imaging agent and methods for diagnosing adisease or disorder in a subject, preferably a mammalian subject, andmost preferably a human subject who is suspected of suffering from amedical disorder, and in particular a cancer, infectious disease,autoimmunity disorder and transplant rejection.

The formulations and modes of administration described herewith for thepharmaceutical compositions can also be suitable to the imaging ordiagnosis compositions (“diagnostic compositions”) according to theinvention.

In a further aspect, the present invention also relates to akit-of-parts comprising at least one of:

-   -   (i) a complex according to the present invention as described        above,    -   (ii) a nucleic acid according to the present invention as        described above,    -   (iii) a vector according to the present invention as described        above,    -   (iv) a host cell according to the present invention as described        above, and    -   (v) a cell loaded with a complex according to the present        invention as described above.

In particular, the kit-of-parts of the invention may comprise more thanone component (i) to (v). For example, the kit-of-parts according to thepresent invention may comprise at least two different complexes under(i), at least two different nucleic acids under (ii), at least twodifferent vectors under (iii), at least two different host cells under(iv), and/or at least two different cells under (v); e.g., thekit-of-parts of the invention may comprise at least two differentcomplexes (i) and/or at least two different nucleic acids (ii).

For example, the different complexes (i) comprised by the kit-of-partsas described above may differ in either component a), i.e. in the cellpenetrating peptides, in component b), i.e. in the antigens or antigenicepitopes or in the subsets of more than one antigen or antigenicepitope, or in component c), i.e. in the TLR peptide agonist or in thesubset of more than one TLR peptide agonist; or the different complexes(i) comprised by the kit-of-parts as described above may differ in twoout of the three components a), b), and c); or the different complexes(i) comprised by the kit-of-parts as described above may differ in allthree components a), b), and c) of the complex. Accordingly, thedifferent nucleic acids (ii) comprised by the kit-of-parts as describedabove may differ in that they encode such different complexes; thedifferent vectors (iii) comprised by the kit-of-parts as described abovemay differ in that they comprise such different nucleic acids; thedifferent host cells (iv) comprised by the kit-of-parts as describedabove may differ in that they comprise such different vectors; and thedifferent cells loaded with a complex (v) comprised by the kit-of-partsas described above may differ in that they are loaded with suchdifferent complexes.

The various components of the kit-of-parts may be packaged in one ormore containers. The above components may be provided in a lyophilizedor dry form or dissolved in a suitable buffer. The kit may also compriseadditional reagents including, for instance, preservatives, growthmedia, and/or buffers for storage and/or reconstitution of theabove-referenced components, washing solutions, and the like. Inaddition, the kit-of-parts according to the present invention mayoptionally contain instructions of use.

Moreover, the present invention also provides a vaccination kit fortreating, preventing and/or stabilizing a cancer or an infectiousdisease, comprising the pharmaceutical composition according to thepresent invention or a vaccine according to the present invention andinstructions for use of said pharmaceutical composition or of saidvaccine.

Thus, the present invention also provides a kit comprising the complexaccording to the present invention as described herein, the cellaccording to the present invention as described herein, the compositionaccording to the present invention as described herein, the vaccineaccording to the present invention as described herein, and/or thepharmaceutical composition according to the present invention asdescribed herein.

Preferably, such a kit further comprises a package insert or instructionleaflet with directions to treat cancer and/or a hematological disorder,preferably a malignant neoplasm of the brain or a malignant neoplasm oflymphoid, hematopoietic and related tissue, most preferably glioblastomaby using the complex according to the present invention as describedherein, the cell according to the present invention as described herein,the composition according to the present invention as described herein,the vaccine according to the present invention as described herein,and/or the pharmaceutical composition according to the present inventionas described herein.

Such a kit may be in particular used in medicine, e.g. in the preventionand/or treatment of cancer.

In addition, the compositions and/or the kit-of-parts according to thepresent inventions may be used in imaging techniques and/or in diagnosisof disease or disorder as disclosed herein.

Use and Methods according to the Invention

In another aspect, the present invention provides the use of any one of:(i) a complex according to the present invention, and/or (ii) cells,such as antigen-presenting cells, loaded with a complex according to thepresent invention, (for the preparation of a medicament) for theprevention, treatment or stabilization of a disease or disorder, such asthose which can be treated by immunotherapy, including cancers,infectious diseases, autoimmunity disorders, hematological diseases andtransplant rejections. Accordingly, the present invention provides anyone of: (i) a complex according to the present invention, and/or (ii)cells, such as antigen-presenting cells, loaded with a complex accordingto the present invention, for use in the prevention, treatment orstabilization of a disease or disorder, such as those which can betreated by immunotherapy, including cancers, infectious diseases,autoimmunity disorders, hematological diseases and transplantrejections.

The present invention also provides a complex according to the presentinvention, which allows the transport and presentation of the at leastone antigen or antigenic epitope comprised by the complex at the cellsurface of antigen presenting cells in an MHC class I and/or MHC classII context, for use in vaccination and/or immunotherapy.

According to another aspect, the present invention provides a method ofpreventing, treating or repressing a disease or disorder such as thosewhich can be treated by immunotherapy, including cancers, infectiousdiseases, autoimmunity disorders, hematological diseases and transplantrejections, wherein said method comprises administering any one of: (i)a complex of the invention, (ii) cells, such as antigen-presentingcells, loaded with a complex of the invention, or (iii) a pharmaceuticalformulation of (i) to (ii), to said subject.

Moreover, the present invention provides a method for eliciting orimproving, in a subject, an immune response against one or multipleepitopes that is dependent on CD4⁺ helper T cells and/or CD8⁺ cytotoxicT cells, wherein said method comprises administering any one of: (i) acomplex according to the present invention, and/or (ii) cells, such asantigen-presenting cells, loaded with said complex, or (iii) apharmaceutical formulation of (i) to (ii), to said subject.

An immune response that is dependent on CD4⁺ and/or CD8⁺ response can bedetermined by evaluating an inflammatory response, a pro-inflammatorycytokine response, including an increase in the expression of one ormore of IFN-γ, TNF-α and IL-2 mRNA or protein relative to the levelbefore administration of the compounds of the invention. It can also bemeasured by an increase in the frequency or absolute number ofantigen-specific T cells after administration of the compounds of theinvention, measured by HLA-peptide multimer staining, ELISPOT assays,and delayed type hypersensitivity tests. It can also be indirectlymeasured by an increase in antigen-specific serum antibodies that aredependent on antigen-specific T helper cells.

The present invention also provides a method for eliciting or improving,in a subject, an immune response against one or multiple antigens orantigenic epitopes that is restricted by multiple MHC class I moleculesand/or multiple MHC class II molecules, wherein said method comprisesadministering any one of: (i) a complex according to the presentinvention, and/or (ii) cells, such as antigen-presenting cells, loadedwith said complex, or (iii) a pharmaceutical formulation of (i) to (ii),to said subject.

A method for eliciting or improving, in a subject, an immune responseagainst multiple epitopes that is restricted by multiple MHC class Imolecules and/or multiple MHC class II molecules can be determined byevaluating a cytokine response, including an increase in the expressionof one or more of IFN-γ, TNF-α and IL-2 mRNA or protein relative to thelevel before administration of the compounds of the invention, after invitro stimulation of T cells with individual peptides binding todiscrete MHC class I and class II molecules on antigen presenting cells.Restriction to different MHC molecules can also be validated by usingantigen presenting cells expressing different MHC molecules, or by usingMHC blocking antibodies. It can also be measured by an increase in thefrequency or absolute number of antigen-specific T cells afteradministration of the compounds of the invention, measured byHLA-peptide multimer staining, which uses multimers assembled withdiscrete MHC molecules.

Preferably, in the methods for eliciting or improving an immune responseagainst one or multiple antigens or antigenic epitopes according to thepresent invention, the immune response is directed against one ormultiple epitopes of a tumor-associated antigen or a tumor-specificantigen as, for instance, a combination of glioma epitopes such as thosedescribed in Novellino et al. (2005, Cancer Immunol Immunother,54(3):187-207) and Vigneron et al. (2013, Cancer Immun.13:15).

Alternatively or additionally, the immune response may be directedagainst multiple epitopes of an antigenic protein from a pathogen.

The methods according to the present invention as described herein, maybe for eliciting or improving, in a subject, an immune response againstone or multiple epitopes that is restricted by MHC class I moleculesand/or MHC class II molecules.

In particular, the present invention thus provides a method for treatingcancer or initiating, enhancing or prolonging an anti-tumor-response ina subject in need thereof comprising administering to the subject acomplex comprising:

-   -   a cell penetrating peptide;    -   at least one antigen or antigenic epitope; and    -   at least one TLR peptide agonist,    -   wherein the components a)-c) are covalently linked.

In such a method it is preferred that the complex according to thepresent invention as described herein, the cell according to the presentinvention as described herein, the composition according to the presentinvention as described herein, the vaccine according to the presentinvention as described herein, and/or the pharmaceutical compositionaccording to the present invention as described herein is administeredto the subject.

Preferably, the subject has cancer and/or was diagnosed with cancer.

Furthermore, the present invention thus provides a method for treatingcancer and/or a hematological disorder, preferably a malignant neoplasmof the brain or a malignant neoplasm of lymphoid, hematopoietic andrelated tissue in a subject in need thereof, most preferablyglioblastoma, comprising administering to the subject a complexcomprising:

-   -   a cell penetrating peptide;    -   at least one antigen or antigenic epitope; and    -   at least one TLR peptide agonist,    -   wherein the components a)-c) are covalently linked.

Thereby, it is preferred that the complex according to the presentinvention as described herein, the cell according to the presentinvention as described herein, the composition according to the presentinvention as described herein, the vaccine according to the presentinvention as described herein, and/or the pharmaceutical compositionaccording to the present invention as described herein is administeredto the subject.

In another aspect, the present invention provides the use of any one of:(i) a complex according to the present invention, and/or (ii) cells,such as antigen-presenting cells, loaded with the complex according tothe present invention, for the preparation of an imaging composition forimaging techniques or for the preparation of a diagnosis composition(“diagnostic compositions”) for diagnosing a disease or disorder,respectively. The diseases or disorders that can be diagnosed with theinvention include those which can be treated by immunotherapy, forinstance cancers, infectious diseases, autoimmunity disorders andtransplant rejections. In particular, the diseases, which can bediagnosed by using a diagnositic composition according to the presentinvention are the diseases, which can be treated and/or prevented byusing a complex according to the present invention as described herein.A diagnostic composition according to the present invention comprises atleast one component selected from:

-   -   (i) a complex according to the present invention as described        above,    -   (ii) a nucleic acid according to the present invention as        described above,    -   (iii) a vector according to the present invention as described        above,    -   (iv) a host cell according to the present invention as described        above, and    -   (v) a cell loaded with a complex according to the present        invention as described above.

Preferably, the diagnostic composition according to the presentinvention comprises the complex according to the present invention asdescribed above.

In particular, the complex according to the present invention, the cell,such as antigen-presenting cell, loaded with the complex according tothe present invention, the inventive composition, the inventivepharmaceutical composition or the inventive vaccine or, most preferably,the inventive diagnostic composition may be utilized in diagnosis as adiagnostic tool, e.g. in (in vivo or in vitro) assays, e.g. inimmunoassays, to detect, prognose, diagnose, or monitor variousconditions and disease states of disorders or diseases mentioned.

As an example, (in vitro) assays may be performed by delivering thecomplex according to the present invention, the cell, such asantigen-presenting cell, loaded with the complex according to thepresent invention, the inventive composition, the inventivepharmaceutical composition or the inventive vaccine or, most preferably,the inventive diagnostic composition to target cells typically selectedfrom e.g. cultured animal cells, human cells or micro-organisms, and tomonitor the cell response by biophysical methods typically known to askilled person. The target cells typically used therein may be culturedcells (in vitro), e.g. cells isolated from human or animal body, such asblood cells isolated from human or animal body, or in vivo cells, i.e.cells composing the organs or tissues of living animals or humans, ormicroorganisms found in living animals or humans. Particularlypreferable in this context are so called markers or labels, which may becontained in the complex according to the present invention and, inparticular, in the diagnostic composition according to the presentinevntion.

According to a further aspect, the invention provides a method ofdiagnosing a disease or disorder in a subject, wherein said methodcomprises administering any one of: (i) a complex of the invention, (ii)cells, such as antigen-presenting cells, loaded with the complex of theinvention, or (iii) a pharmaceutical formulation of (i) to (ii), to saidsubject or to said subject's sample ex vivo.

According to another aspect, the present invention provides an imagingmethod wherein said method comprises using, in vitro, ex vivo or invivo, any one of: (i) a complex of the invention, (ii) cells, such asantigen-presenting cells, loaded with the complex of the invention, or(iii) a pharmaceutical formulation of (i) to (ii).

Preferably, uses and methods according to the present invention compriseadministration of a complex according to the invention.

Moreover, uses and methods according to the present invention compriseadministration of more than one complex, cells, or pharmaceuticalformulation according to the invention. For example, in the uses andmethods according to the present invention, at least two differentcomplexes are used or administered, wherein each complex comprises atleast one antigen or antigenic epitope and said antigen or antigenicepitope or (if more than one antigen or antigenic epitope is comprisedby said complex) said subset of antigens or antigenic epitopes aredifferent between the two complexes.

For example, the different complexes (i) comprised by the composition asdescribed above may differ in either component a), i.e. in the cellpenetrating peptides, in component b), i.e. in the antigens or antigenicepitopes or in the subsets of more than one antigen or antigenicepitope, or in component c), i.e. in the TLR peptide agonist or in thesubset of more than one TLR peptide agonist; or the different complexes(i) comprised by the composition as described above may differ in twoout of the three components a), b), and c); or the different complexes(i) comprised by the composition as described above may differ in allthree components a), b), and c) of the complex. Accordingly, thedifferent nucleic acids (ii) comprised by the composition as describedabove may differ in that they encode such different complexes; thedifferent vectors (iii) comprised by the composition as described abovemay differ in that they comprise such different nucleic acids; thedifferent host cells (iv) comprised by the composition as describedabove may differ in that they comprise such different vectors; and thedifferent cells loaded with a complex (v) comprised by the compositionas described above may differ in that they are loaded with suchdifferent complexes.

Moreover, in the uses and methods according to the present invention,the cells according to the present invention may be antigen presentingcells, in particular dendritic cells, more preferably dendritic cellsfrom the subject to be treated.

Diseases to be Treated or Prevented

The term “disease” as used in the context of the present invention isintended to be generally synonymous, and is used interchangeably with,the terms “disorder” and “condition” (as in medical condition), in thatall reflect an abnormal condition of the human or animal body or of oneof its parts that impairs normal functioning, is typically manifested bydistinguishing signs and symptoms, and causes the human or animal tohave a reduced duration or quality of life.

Diseases to be treated and/or prevented by use of the complex accordingto the present invention; the cell, such as antigen-presenting cell,loaded with the complex according to the present invention; theinventive composition; the inventive pharmaceutical composition or theinventive vaccine include cancer, hematological disorders, infectiousdiseases, autoimmunity disorders and transplant rejections. Thereby,treatment and/or prevention of cancer and infectious diseases ispreferred and treatment and/or prevention of cancer is more preferred.For cancer, a malignant neoplasm of the brain or a malignant neoplasm oflymphoid, hematopoietic and related tissue are preferred andglioblastoma is more preferred.

Preferably, the complex according to the present invention; the cell,such as antigen-presenting cell, loaded with the complex according tothe present invention; the inventive composition; the inventivepharmaceutical composition or the inventive vaccine, may be used for(the preparation of a medicament for) the prophylaxis, treatment and/oramelioration of cancer or tumor diseases, including diseases caused bydefective apoptosis, preferably selected from acusticus neurinoma, analcarcinoma, astrocytoma, basalioma, Behcet's syndrome, bladder cancer,blastomas, bone cancer, brain metastases, brain tumors, brain cancer(glioblastomas), breast cancer (mamma carcinoma), Burkitt's lymphoma,carcinoids, cervical cancer, colon carcinoma, colorectal cancer, corpuscarcinoma, craniopharyngeomas, CUP syndrome, endometrial carcinoma, gallbladder cancer, genital tumors, including cancers of the genitourinarytract, glioblastoma, gliomas, head/neck tumors, hepatomas, histocyticlymphoma, Hodgkin's syndromes or lymphomas and non-Hodgkin's lymphomas,hypophysis tumor, intestinal cancer, including tumors of the smallintestine, and gastrointestinal tumors, Kaposi's sarcoma, kidney cancer,kidney carcinomas, laryngeal cancer or larynx cancer, leukemia,including acute myeloid leukaemia (AML), erythroleukemia, acute lymphoidleukaemia (ALL), chronic myeloid leukaemia (CML), and chroniclymphocytic leukaemia (CLL), lid tumor, liver cancer, liver metastases,lung carcinomas (=lung cancer=bronchial carcinoma), small cell lungcarcinomas and non-small cell lung carcinomas, and lung adenocarcinoma,lymphomas, lymphatic cancer, malignant melanomas, mammary carcinomas(=breast cancer), medulloblastomas, melanomas, meningiomas, Mycosisfungoides, neoplastic diseases neurinoma, oesophageal cancer,oesophageal carcinoma (=oesophageal cancer), oligodendroglioma, ovariancancer (=ovarian carcinoma), ovarian carcinoma, pancreatic carcinoma(=pancreatic cancer), penile cancer, penis cancer, pharyngeal cancer,pituitary tumour, plasmocytoma, prostate cancer (=prostate tumors),rectal carcinoma, rectal tumors, renal cancer, renal carcinomas,retinoblastoma, sarcomas, Schneeberger's disease, skin cancer, e.g.melanoma or non-melanoma skin cancer, including basal cell and squamouscell carcinomas as well as psoriasis, pemphigus vulgaris, soft tissuetumours, spinalioma, stomach cancer, testicular cancer, throat cancer,thymoma, thyroid carcinoma, tongue cancer, urethral cancer, uterinecancer, vaginal cancer, various virus-induced tumors such as, forexample, papilloma virus-induced carcinomas (e.g. cervicalcarcinoma=cervical cancer), adenocarcinomas, herpes virus-induced tumors(e.g. Burkitt's lymphoma, EBV-induced B-cell lymphoma, cervixcarcinoma), heptatitis B-induced tumors (hepatocell carcinomas), HTLV-1-and HTLV-2-induced lymphomas, vulval cancer, wart conditions orinvolvement, etc. In the present context, the terms “therapy” and“therapeutic” preferably mean to have at least some minimalphysiological effect upon being administered to a living body. Forexample, a physiological effect upon administering a “therapeutic”anti-tumor compound may be the inhibition of tumor growth, or decreasein tumor size, or prevention reoccurrence of the tumor. Preferably, inthe treatment of cancer or neoplastic disease, a compound which inhibitsthe growth of a tumor or decreased the size of the tumor or prevents thereoccurrence of the tumor would be considered therapeutically effective.The term “anti-tumor drug” therefore preferably means any therapeuticagent having therapeutic effect against a tumor, neoplastic disease orcancer.

Examples of cancers include brain cancer, prostate cancer, breastcancer, ovarian cancer, esophageal cancer, lung cancer, liver cancer,kidney cancer, melanoma, gut carcinoma, lung carcinoma, head and necksquamous cell carcinoma, chronic myeloid leukemia, colorectal carcinoma,gastric carcinoma, endometrial carcinoma, myeloid leukemia, lungsquamous cell carcinoma, acute lymphoblastic leukemia, acute myelogenousleukemia, bladder tumor, promyelocytic leukemia, non-small cell lungcarcinoma, sarcoma.

The cancer may be a solid tumor, blood cancer, or lymphatic cancer. Thecancer may be benign or metastatic.

Preferably, the cancer to be prevented and/or treated is a glioma, morepreferably highly invasive glioblastoma multiforme (GBM). Gliomas arethe most frequent form of primary brain tumors in adults, withglioblastoma multiforme (GBM) having the poorest prognosis. This tumoris notorious for its highly invasive and aggressive behavior. Inparticular, the complex according to the present invention; the cell,such as antigen-presenting cell, loaded with the complex according tothe present invention; the inventive composition; the inventivepharmaceutical composition or the inventive vaccine may be used inconjunction with existing modalities for glioma, more specificallyhighly invasive GBM. T lymphocytes can actively seek out neoplasticcells in the brain, and have the potential to safely eliminate specifictumor cells without damaging the surrounding healthy tissues.

Moreover, the complex according to the present invention; the cell, suchas antigen-presenting cell, loaded with the complex according to thepresent invention; the inventive composition; the inventivepharmaceutical composition or the inventive vaccine, may be used for(the preparation of a medicament for) the prophylaxis, treatment and/oramelioration of infectious diseases, preferably viral, retroviral,bacterial or protozoological infectious diseases. Such infectiousdiseases are typically selected from AIDS, anthrax, Japaneseencephalitis, bacterial infectious diseases such as miscarriage(prostate inflammation), anthrax, appendicitis, borreliosis, botulism,Camphylobacter, Chlamydia trachomatis (inflammation of the urethra,conjunctivitis), cholera, diphtheria, donavanosis, epiglottitis, typhusfever, gas gangrene, gonorrhoea, rabbit fever, Heliobacter pylori,whooping cough, climatic bubo, osteomyelitis, Legionnaire's disease,chicken-pox, condyloma acuminata, cytomegalic virus (CMV), dengue fever,early summer meningoencephalitis (ESME), Ebola virus, colds, fifthdisease, foot-and-mouth disease, herpes simplex type I, herpes simplextype II, herpes zoster, HSV, infectious diseases caused by parasites,protozoa or fungi, such as amoebiasis, bilharziosis, Chagas disease,Echinococcus, fish tapeworm, fish poisoning (Ciguatera), fox tapeworm,athlete's foot, canine tapeworm, candidosis, yeast fungus spots,scabies, cutaneous Leishmaniosis, lambliasis (giardiasis), lice,onchocercosis (river blindness), fungal diseases, bovine tapeworm,schistosomiasis, porcine tapeworm, toxoplasmosis, trichomoniasis,trypanosomiasis (sleeping sickness), visceral Leishmaniosis,nappy/diaper dermatitis or miniature tapeworm, infectious erythema,influenza, Kaposi's sarcoma, Lassa fever, Leishmaniasis, leprosy,listeriosis, Lyme borreliosis, malaria, Marburg virus infection,measles, meningitis, including bacterial meningitis, molluscumcontagiosum, mononucleosis, mumps, Mycoplasma hominis, neonatal sepsis(Chorioamnionitis), noma, Norwalk virus infection, otitis media,paratyphus, Pfeiffer's glandular fever, plague, pneumonia, polio(poliomyelitis, childhood lameness), pseudo-croup, rabies, Reiter'ssyndrome, Rocky Mountain spotted fever, Salmonella paratyphus,Salmonella typhus, SARS, scarlet fever, shingles, hepatitis, smallpox,soft chancre, syphilis, tetanus,three-day fever, tripper, tsutsugamushidisease, tuberculosis, typhus, vaginitis (colpitis), viral diseasescaused by cytomegalovirus (CMV), orthopox variola virus, orthopoxalastrim virus, parapox ovis virus, molluscum contagiosum virus, herpessimplex virus 1, herpes simplex virus 2, herpes B virus, varicellazoster virus, pseudorabies virus, human cytomegaly virus, human herpesvirus 6, human herpes virus 7, Epstein-Barr virus, human herpes virus 8,hepatitis B virus, chikungunya virus, O′nyong'nyong virus, rubivirus,hepatitis C virus, GB virus C, West Nile virus, dengue virus, yellowfever virus, louping ill virus, St. Louis encephalitis virus, Japan Bencephalitis virus, Powassan virus, FSME virus, SARS, SARS-associatedcorona virus, human corona virus 229E, human corona virus Oc43,Torovirus, human T cell lymphotropic virus type I, human T celllymphotropic virus type II, HIV (AIDS), i.e. human immunodeficiencyvirus type 1 or human immunodeficiency virus type 2, influenza virus,Lassa virus, lymphocytic choriomeningitis virus, Tacaribe virus, Juninvirus, Machupo virus, Borna disease virus, Bunyamwera virus, Californiaencephalitis virus, Rift Valley fever virus, sand fly fever virus,Toscana virus, Crimean-Congo haemorrhagic fever virus, Hazara virus,Khasan virus, Hantaan virus, Seoul virus, Prospect Hill virus, Puumalavirus, Dobrava Belgrade virus, Tula virus, sin nombre virus, LakeVictoria Marburg virus, Zaire Ebola virus, Sudan Ebola virus, IvoryCoast Ebola virus, influenza virus A, influenza virus B, influenzaviruses C, parainfluenza virus, measles virus, mumps virus, respiratorysyncytial virus, human metapneumovirus, vesicular stomatitis Indianavirus, rabies virus, Mokola virus, Duvenhage virus, European batlyssavirus 1+2, Australian bat lyssavirus, adenoviruses A-F, humanpapilloma viruses, condyloma virus 6, condyloma virus 11, polyomaviruses, adeno-associated virus 2, rotaviruses, or orbiviruses,Varicella including Varizella zoster, and malaria parasite (Plasmodiumfalciparum, Plasmodium vivax, Plasmodium ovale, Plasmodium malariae,Plasmodium knowlesi), viral infectious diseases such as AIDS, infectiousdiseases caused by Condyloma acuminata, hollow warts, Dengue fever,three-day fever, Ebola virus, cold, early summer meningoencephalitis(FSME), flu, shingles, hepatitis, herpes simplex type I, herpes simplextype II, Herpes zoster, influenza, Japanese encephalitis, Lassa fever,Marburg virus, warts, West Nile fever, yellow fever, etc.

Examples of infectious diseases include diseases caused by viruses,bacteria, fungi, protozoa and multicellular parasites. They include, forinstance, Amoebiasis, Anthrax, Buruli Ulcer (Mycobacterium ulcerans),Caliciviruses associated diarrhoea, Campylobacter diarrhoea, CervicalCancer (Human papillomavirus), Chlamydia trachomatis associated genitaldiseases, Cholera, Crimean-Congo haemorrhagic fever, Dengue Fever,Diptheria, Ebola haemorrhagic fever, Enterotoxigenic Escherichia coli(ETEC) diarrhoea, Gastric Cancer (Helicobacter pylori), Gonorrhea, GroupA Streptococcus associated diseases, Group B Streptococcus associateddiseases, Haemophilus influenzae B pneumonia and invasive disease,Hepatitis A, Hepatitis B, Hepatitis C, Hepatitis E diarrhoea, Herpessimplex type 2 genital ulcers, HIV/AIDS, Hookworm Disease, Influenza,Japanese encephalitis, Lassa Fever, Leishmaniasis, Leptospirosi, Livercancer (Hepatitis B), Liver Cancer (Hepatitis C), Lyme Disease, Malaria,Marburg haemorrhagic fever, Measles, Mumps, Nasopharyngeal cancer(Epstein-Barr virus), Neisseria meningitidis Meningitis, Parainfluenzaassociated pneumonia, Pertussis, Plague, Poliomyelitis, Rabies,Respiratory syncytial virus (RSV) pneumonia, Rift Valley fever,Rotavirus diarrhoea, Rubella, Schistosomiasis, Severe Acute RespiratorySyndrome (SARS), Shigellosis, Smallpox, Staphylococcus aureus associateddiseases, Stomach Cancer (Helicobacter pylori), Streptococcus pneumoniaeand invasive disease, Tetanus, Tick-borne encephalitis, Trachoma,Tuberculosis, Tularaemia, Typhoid fever, West-Nile virus associateddisease, Yellow fever.

Moreover, the complex according to the present invention; the cell, suchas antigen-presenting cell, loaded with the complex according to thepresent invention; the inventive composition; the inventivepharmaceutical composition or the inventive vaccine, may be used for(the preparation of a medicament for) the prophylaxis, treatment and/oramelioration of autoimmune disorders, for example autoimmune diseases ofthe CNS, auto-inflammatory diseases, Celiac disease; Sjogren's syndrome,systemic lupus erythematosus etc. Typically, autoimmune diseases arisefrom an abnormal immune response of the body against substances andtissues normally present in the body (autoimmunity). This may berestricted to certain organs (e.g. in autoimmune thyroiditis) or mayinvolve a particular tissue in different places (e.g. Goodpasture'sdisease which may affect the basement membrane in both the lung and thekidney). Autoimmune diseases may be classified by corresponding type ofhypersensitivity: type I (i.e. urticaria induced by autologous serum),type II, type III, or type IV.

Examples of autoimmune diseases include Blau syndrome, Bullouspemphigoid, Cancer, Castleman's disease, Celiac disease, Chagas disease,Chronic inflammatory demyelinating polyneuropathy, Chronic recurrentmultifocal osteomyelitis, chronic obstructive pulmonary disease,Churg-Strauss syndrome, Cicatricial pemphigoid, Cogan syndrome, Coldagglutinin disease, Complement component 2 deficiency, Contactdermatitis, Cranial arteritis, CREST syndrome, Crohn's disease,Cushing's Syndrome, Dercum's disease, Dermatitis herpetiformis,Dermatomyositis, Diabetes mellitus type 1, Diffuse cutaneous systemicsclerosis, Dressler's syndrome, lupus, Discoid lupus erythematosus,Eczema, Acute disseminated encephalomyelitis (ADEM), Addison's disease,Agammaglobulinemia, Amyotrophic lateral sclerosis (Also Lou Gehrig'sdisease; Motor Neuron Disease), Ankylosing Spondylitis Antiphospholipidsyndrome, Antisynthetase syndrome, Atopic dermatitis, Autoimmuneaplastic anemia, Autoimmune cardiomyopathy, Autoimmune hemolytic anemia,Autoimmune hepatitis, Autoimmune inner ear disease, Autoimmunelymphoproliferative syndrome, Autoimmune peripheral neuropathy,Autoimmune pancreatitis, Autoimmune polyendocrine syndrome, Autoimmuneprogesterone dermatitis, Autoimmune thrombocytopenic purpura, Autoimmuneurticarial, Autoimmune uveitis, Balo disease/Balo concentric sclerosis,Behcet's disease, Berger's disease, Bickerstaff's encephalitis,Endometriosis, Enthesitis-related arthritis, Eosinophilicgastroenteritis, Epidermolysis bullosa acquisita, Erythroblastosisfetalis, Evan's syndrome, Fibrodysplasia ossificans, Fibrosingalveolitis (or Idiopathic pulmonary fibrosis), Gastritis,Glomerulonephritis, Goodpasture's syndrome, Graves' disease,Guillain-Barre syndrome, Hashimoto's encephelopathy, Hashimoto'sthyroiditis, Gestational Pemphigoid, Hidradenitis suppurativa,Hypogammaglobulinemia, Idiopathic thrombocytopenic purpura (Autoimmunethrombocytopenic purpura), IgA nephropathy, Occular cicatricialpemphigoid, Inclusion body myositis, Rheumatoid arthritis, Chronicinflammatory Rheumatic fever, demyelinating polyneuropathy, Sarcoidosis,Palindromic rheumatism, Interstitial cystitis, Juvenile idiopathicSchizophrenia, PANDAS (pediatric arthritis aka Juvenile autoimmunerheumatoid arthritis), Schmidt syndrome, neuropsychiatric Kawasaki'sdisease another form of APS, Schnitzler syndrome, Paraneoplasticcerebellar myasthenic syndrome, Leukocytoclastic Serum Sickness, Lichenplanus, Sjögren's syndrome, Lichen sclerosus, Parsonage-Tumer, LinearIgA disease, Still's disease, Pemphigus vulgaris, Lupoid hepatitis,Autoimmune hepatitis, Stiff person syndrome, Pernicious anaemia,Subacute bacterial endocarditis (SBE), POEMS syndrome, Lupuserythematosus, Sweet's syndrome, Sympathetic ophthalmia, Meniere'sdisease, Systemic lupus, Primary biliary cirrhosis, Miller-Fishersyndrome, Takayasu's arteritis, cholangitis, Progressive inflammatoryneuropathy, Mucha-Habermann disease, Psoriasis, Psoriatic arthritis,Pyoderma gangrenosum, Multiple sclerosis, Pure red cell aplasia,Rasmussen's encephalitis, Myasthenia gravis, Transverse myelitis,Raynaud phenomenon, Microscopic colitis, Ulcerative colitis, Myositis,idiopathic inflammatory bowel disease (IBD), Neuromyelitis optica,Devic's disease, and Neuromyotonia.

Moreover, the complex according to the present invention; the cell, suchas antigen-presenting cell, loaded with the complex according to thepresent invention; the inventive composition; the inventivepharmaceutical composition or the inventive vaccine, may be used for(the preparation of a medicament for) the prophylaxis, treatment and/oramelioration of hematological disorders, which are typically disorderswhich primarily affect the blood. Thereby, Hematological malignanciesare preferred.

Examples of hematological diseases include myeloid disorders, includingHemoglobinopathies (congenital abnormality of the hemoglobin molecule orof the rate of hemoglobin synthesis), e.g. Sickle-cell disease,Thalassemia, Methemoglobinemia; Anemias (lack of red blood cells orhemoglobin), e.g. Iron deficiency anemia, Megaloblastic anemia includingVitamin B₁₂ deficiency, Pernicious anemia, and Folate deficiency,Hemolytic anemias (destruction of red blood cells) including Geneticdisorders of RBC membrane such as Hereditary spherocytosis, Hereditaryelliptocytosis, and Congenital dyserythropoietic anemia, Geneticdisorders of RBC metabolism such as Glucose-6-phosphate dehydrogenasedeficiency (G6PD), and Pyruvate kinase deficiency, Immune mediatedhemolytic anemia (direct Coombs test is positive) such as Autoimmunehemolytic anemia including Warm antibody autoimmune hemolytic anemia(such as Idiopathic, Systemic lupus erythematosus (SLE), and Evans'syndrome (antiplatelet antibodies and hemolytic antibodies)) and Coldantibody autoimmune hemolytic anemia (such as Idiopathic coldhemagglutinin syndrome, Infectious mononucleosis, and Paroxysmal coldhemoglobinuria), Alloimmune hemolytic anemia including Hemolytic diseaseof the newborn (HDN) (such as Rh disease (Rh D), ABO hemolytic diseaseof the newborn, Anti-Kell hemolytic disease of the newborn, Rhesus chemolytic disease of the newborn, Rhesus E hemolytic disease of thenewborn, and other blood group incompatibility (RhC, Rhe, Kid, Duffy,MN, P and others)), Drug induced immune mediated hemolytic anemiaincluding Penicillin (high dose) and Methyldopa, Hemoglobinopathies(i.e. where these is an unstable or crystalline hemoglobin), Paroxysmalnocturnal hemoglobinuria (rare acquired clonal disorder of red bloodcell surface proteins), Direct physical damage to RBCs includingMicroangiopathic hemolytic anemia and Secondary to artificial heartvalve(s), Aplastic anemia such as Fanconi anemia, Diamond-Blackfananemia (inherited pure red cell aplasia), and Acquired pure red cellaplasia; Decreased numbers of cells, e.g. Myelodysplastic syndrome,Myelofibrosis, Neutropenia (decrease in the number of neutrophils),Agranulocytosis, Glanzmann's thrombasthenia, and Thrombocytopenia(decrease in the number of platelets) including Idiopathicthrombocytopenic purpura (ITP), Thrombotic thrombocytopenic purpura(TTP), and Heparin-induced thrombocytopenia (HIT); Myeloproliferativedisorders (Increased numbers of cells), e.g. Polycythemia vera (increasein the number of cells in general), Erythrocytosis (increase in thenumber of red blood cells), Leukocytosis (increase in the number ofwhite blood cells), Thrombocytosis (increase in the number ofplatelets), and Myeloproliferative disorder; Coagulopathies (disordersof bleeding and coagulation), e.g. Thrombocytosis, Recurrent thrombosis,Disseminated intravascular coagulation, Disorders of clotting proteinsincluding Hemophilia such as Hemophilia A, Hemophilia B (also known asChristmas disease), and Hemophilia C, Von Willebrand disease,Disseminated intravascular coagulation, Protein S deficiency, andAntiphospholipid syndrome, and Disorders of platelets includingThrombocytopenia, Glanzmann's thrombasthenia, and Wiskott-Aldrichsyndrome. Moreover, examples of hematological diseases also includeHematological malignancies including Hematological malignancies, e.g.Lymphomas including Hodgkin's disease and Non-Hodgkin's lymphoma such asBurkitt's lymphoma, Anaplastic large cell lymphoma, Splenic marginalzone lymphoma, Hepatosplenic T-cell lymphoma, and AngioimmunoblasticT-cell lymphoma (AILT), Myelomas such as Multiple myeloma, Waldenströmmacroglobulinemia, and Plasmacytoma, Leukemias such as Acute lymphocyticleukemia (ALL), Chronic lymphocytic leukemia (CLL), Acute myelogenousleukemia (AML), Chronic Idiopathic Myleofibrosis (MF), Chronicmyelogenous leukemia (CML), T-cell prolymphocytic leukemia (T-PLL),B-cell prolymphocytic leukemia (B-PLL), Chronic neutrophilic leukemia(CNL), Hairy cell leukemia (HCL), T-cell large granular lymphocyteleukemia (T-LGL), and Aggressive NK-cell leukemia. Moreover, examples ofhematological diseases also include miscellaneous haematologicaldiseases including Hemochromatosis, Asplenia, Hypersplenism such asGauchers disease, Monoclonal gammopathy of undetermined significance,Hemophagocytic lymphohistiocytosis, and Tempi syndrome. Moreover,examples of hematological diseases also include Hematological changessecondary to non-hematological disorders including Anemia of chronicdisease, Infectious mononucleosis, AIDS, Malaria, and Leishmaniasis.

Moreover, the complex according to the present invention; the cell, suchas antigen-presenting cell, loaded with the complex according to thepresent invention; the inventive composition; the inventivepharmaceutical composition or the inventive vaccine, may be used for(the preparation of a medicament for) the prophylaxis, treatment and/oramelioration of transplant rejection, including e.g. graft-versus-hostreaction. Transplant rejection includes hyperacute rejection, acuterejection and chronic rejection of a transplant. Examples of transplantrejection include skin, kidney, heart, lung, pancreas, liver, bloodcell, bone marrow, cornea, accidental severed limb, in particularfingers, hand, foot, face, nose, bone, cardiac valve, blood vessel orintestine transplant rejection reaction.

Mode of Administration

The complex according to the present invention; the cell, such asantigen-presenting cell, loaded with the complex according to thepresent invention; the inventive composition; the inventivepharmaceutical composition or the inventive vaccine may be administeredin any manner as described above, including orally, parenterally,intravenously, rectally, or combinations thereof. Parenteraladministration includes, but is not limited to, intravenous,intra-arterial, intra-peritoneal, subcutaneous, intradermal andintramuscular. The complex according to the present invention; the cell,such as antigen-presenting cell, loaded with the complex according tothe present invention; the inventive composition; the inventivepharmaceutical composition or the inventive vaccine may also bepreferably administered via topical, intratumoral, intradermal,subcutaneous, intramuscular, intranasal, or intranodal route. Thecomplex according to the present invention; the cell, such asantigen-presenting cell, loaded with the complex according to thepresent invention; the inventive composition; the inventivepharmaceutical composition or the inventive vaccine may also beadministered in the form of an implant, which allows slow release of thecompositions as well as a slow controlled i.v. infusion. For example,the complex according to the present invention; the cell, such asantigen-presenting cell, loaded with the complex according to thepresent invention; the inventive composition; the inventivepharmaceutical composition or the inventive vaccine may be administeredsubcutaneously.

The administration of complex according to the present invention; thecell, such as antigen-presenting cell, loaded with the complex accordingto the present invention; the inventive composition; the inventivepharmaceutical composition or the inventive vaccine may require multiplesuccessive injections. Thus, the administration may be repeated at leasttwo times, for example once as primary immunization injections and,later, as booster injections.

In particular, the complex according to the present invention; the cell,such as antigen-presenting cell, loaded with the complex according tothe present invention; the inventive composition; the inventivepharmaceutical composition or the inventive vaccine may be administeredrepeatedly or continuously. The complex according to the presentinvention; the cell, such as antigen-presenting cell, loaded with thecomplex according to the present invention; the inventive composition;the inventive pharmaceutical composition or the inventive vaccine may beadministered repeatedly or continuously for a period of at least 1, 2,3, or 4 weeks; 2, 3, 4, 5, 6, 8, 10, or 12 months; or 2, 3, 4, or 5years.

Moreover, the cell penetrating peptide, components a), b), and c), i.e.the at least one antigen or antigenic epitope and the at least one TLRpeptide agonist, composing the complex according to the presentinvention may be contained in separate compositions which are mixed justbefore administration or which are administered simultaneously to thesubject in need thereof.

According to one approach, the complex according to the presentinvention; the cell, such as antigen-presenting cell, loaded with thecomplex according to the present invention; the inventive composition;the inventive pharmaceutical composition or the inventive vaccine may beadministered directly to a patient using the administration routes asdescribed above, in particular for pharmaceutical compositions.Alternatively, the complex according to the present invention; the cell,such as antigen-presenting cell, loaded with the complex according tothe present invention; the inventive composition; the inventivepharmaceutical composition or the inventive vaccine may be administeredto a patient using an ex vivo approach, e.g. by introducing thepharmaceutical composition, the vaccine or the inventive transportercargo conjugate molecule as defined above into cells, preferablyautologous cells, i.e. cells derived from the patient to be treated, andtransplanting these cells into the site of the patient to be treated,optionally subsequent to storing and/or culturing these cells prior totreatment.

The dosage administered, as single or multiple doses, to an individualwill vary depending upon a variety of factors, including pharmacokineticproperties, subject conditions and characteristics (sex, age, bodyweight, health, size), extent of symptoms, concurrent treatments,frequency of treatment and the effect desired.

Typically, for cancer treatment, the therapeutically effective dose of acomplex according to the present invention is from about 0.01 mg to 5 mgper injection, in particular from about 0.1 mg to 2 mg per injection, orfrom about 0.01 nmol to 1 mmol per injection, in particular from 1 nmolto 1 mmol per injection, preferably from 1 μmol to 1 mmol per injection.

Typically, for cancer treatment, the therapeutically effective dose ofan antigen presenting cell loaded with a complex according to thepresent invention is from about 0.2 million cells to 2 million cells perinjection.

Combination Therapy

The administration of the complex according to the present invention;the cell, such as antigen-presenting cell, loaded with the complexaccording to the present invention; the inventive composition; theinventive pharmaceutical composition or the inventive vaccine in themethods and uses according to the invention can be carried out alone orin combination with a co-agent useful for treating and/or stabilizingthe disease or disorder to be treated or repressed.

For instance, in the case of treatment, prevention, or stabilization ofa cancer, the administration of the pharmaceutical compositions in themethods and uses according to the invention can be carried out incombination with substances used in conventional chemotherapy directedagainst solid tumors and for control of establishment of metastases orany other molecule that act by triggering programmed cell death e.g. forexample a co-agent selected from Tumor Necrosis Family Membersincluding, but not limited, to Fas Ligand and tumor necrosis factor(TNF)-related apoptosis inducing (TRAIL) ligand. According to a furtherembodiment, the administration of the complex according to the presentinvention; the cell, such as antigen-presenting cell, loaded with thecomplex according to the present invention; the inventive composition;the inventive pharmaceutical composition or the inventive vaccine in themethods and uses according to the present invention can be carried outin parallel of radiotherapy.

The invention encompasses the administration of the complex according tothe present invention; the cell, such as antigen-presenting cell, loadedwith the complex according to the present invention; the inventivecomposition; the inventive pharmaceutical composition or the inventivevaccine, wherein it is administered to a subject prior to,simultaneously or sequentially with other therapeutic regimens orco-agents useful for treating, and/or stabilizing a cancer and/orpreventing cancer relapsing (e.g. multiple drug regimens), in atherapeutically effective amount. Said complex, cell, composition,vaccine or pharmaceutical composition, that is administeredsimultaneously with said co-agents can be administered in the same ordifferent composition(s) and by the same or different route(s) ofadministration.

Said other therapeutic regimens or co-agents may be selected from thegroup consisting of radiation therapy, chemotherapy, surgery, targetedtherapy (including small molecules, peptides and monoclonal antibodies),and anti-angiogenic therapy. Anti-angiogenic therapy is defined hereinas the administration of an agent that directly or indirectly targetstumor-associated vasculature.

Accordingly, the present invention provides a pharmaceutical formulationcomprising a complex of the invention or a cell of the invention, inparticular an antigen-presenting cell of the invention, combined with atleast one co-agent useful for treating and/or stabilizing a cancerand/or preventing a cancer relapsing, and at least one pharmaceuticallyacceptable carrier.

Moreover, the complex according to the present invention; the cell, suchas antigen-presenting cell, loaded with the complex according to thepresent invention; the inventive composition; the inventivepharmaceutical composition or the inventive vaccine can be administeredafter surgery where solid tumors have been removed as a prophylaxisagainst relapsing and/or metastases.

Moreover, the administration of the imaging or diagnosis composition inthe methods and uses according to the invention can be carried out aloneor in combination with a co-agent useful for imaging and/or diagnosingthe suspected disease or disorder.

Subjects

The present invention can be applied to any subject suffering from anydisease or disorder, depending on the specificity in particular of theat least one antigen or antigenic epitope comprised by the complexaccording to the present invention. In particular, the therapeuticeffect of said complex may be to elicit an immune response directedagainst said antigens or antigenic epitopes, in particular a responsethat is dependent on CD4⁺ helper T cells and/or CD8⁺ cytotoxic T cellsand/or that is restricted by MHC class I molecules and/or MHC class IImolecules.

Preferably, subjects according to the invention are subjects sufferingfrom a cancer, for instance from a cancer of the brain, colon, head orneck, or from a cervical cancer. More preferably, subjects according tothe invention are subjects suffering from a brain cancer includingglioma.

It is also preferred that subjects according to the invention have beensubjected to a surgical removal of a tumor.

Alternatively, subjects according to the invention are subjectssuffering from an infectious disease.

The present invention is not to be limited in scope by the specificembodiments described herein. Indeed, various modifications of theinvention in addition to those described herein will become apparent tothose skilled in the art from the foregoing description and accompanyingfigures. Such modifications fall within the scope of the appendedclaims.

All references cited herein are herewith incorporated by reference.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety. In the case of conflict, the presentspecification, including definitions, will control. In addition, thematerials, methods, and examples are illustrative only and not intendedto be limiting.

BRIEF DESCRIPTION OF THE FIGURES

In the following a brief description of the appended figures will begiven. The figures are intended to illustrate the present invention inmore detail. However, they are not intended to limit the subject matterof the invention in any way.

FIG. 1 shows for Example 1 expression of activation marker CD40 by humanblood monocyte-derived dendritic cells (DCs) from one single buffy. TheDCs were stimulated with 300 nM of EDAZ13Mad5, Z13Mad5, Mad5 or 25 ng/mlof LPS during 48 h. Isotype staining for each condition was alsoperformed (isotype is not shown in the FIG. 1) (one experiment).

FIG. 2 shows for Example 1 expression of activation marker CD86 by humanblood monocyte-derived dendritic cells (DCs) from one single buffy. TheDCs were stimulated with 300 nM of EDAZ13Mad5, Z13Mad5, Mad5 or 25 ng/mlof LPS during 48 h. Isotype staining for each condition was alsoperformed (isotype is not shown in the FIG. 2) (one experiment).

FIG. 3 shows for Example 1 expression of activation marker HLADR byhuman blood monocyte-derived dendritic cells (DCs) from one singlebuffy. The DCs were stimulated with 300 nM of EDAZ13Mad5, Z13Mad5, Mad5or 25 ng/ml of LPS during 48 h. Isotype staining for each condition wasalso performed (isotype is not shown in the FIG. 3) (one experiment).

FIG. 4 shows for Example 1 expression of activation marker CD83 by humanblood monocyte-derived dendritic cells (DCs) from one single buffy. TheDCs were stimulated with 300 nM of EDAZ13Mad5, Z13Mad5, Mad5 or 25 ng/mlof LPS during 48 h. Isotype staining for each condition was alsoperformed (isotype is not shown in the FIG. 4) (one experiment).

FIG. 5 shows for Example 2 functional MHC class I-restrictedcross-presentation in a murine in an vitro system using bone marrowderived dendritic cells (BMDCs) and splenocytes from different TCRtransgenic mice. To this end, BMDCs were loaded overnight with 300 nM ofEDAZ13Mad5, EDAMad5 or Mad5. Efficient MHC class I-restrictedpresentation of OVACD8 epitope and gp100 epitope was monitored after 4days with CFSE-labeled OT1 cells and P-Mel cells respectively. EfficientMHC class II-restricted presentation of OVACD4 epitope was monitoredafter 4 days with CFSE-labeled OT2 cells. As control, BMDCs were pulsedfor 1 h with 5 uM peptide (one experiment representative of 2 individualexperiments).

FIG. 6 shows the results for the 2 nmol groups for Example 3. C57BL/6mice were vaccinated twice (Wk0 and Wk2) with 2 nmol of EDAMad5 orEDAZ13Mad5. Positive control group was vaccinated with Mad5 and MPLA(equimolar to EDA). Mice were bled 7 days after last vaccination andpentamer staining was performed (3-4 mice per group, one experiment).

FIG. 7 shows the results for the 10 nmol groups for Example 3. C57BL/6mice were vaccinated twice (Wk0 and Wk2) with 10 nmol of EDAMad5 orEDAZ13Mad5. Positive control group was vaccinated with Mad5 and MPLA(equimolar to EDA). Mice were bled 7 days after last vaccination andpentamer staining was performed (3-4 mice per group, one experiment).

FIG. 8 shows for Example 3 the percentage of pentamer positive CD8+ Tcells for all groups tested. C57BL/6 mice were vaccinated twice (Wk0 andWk2) with 2 nmol or 10 nmol of EDAMad5 or EDAZ13Mad5. Positive controlgroup was vaccinated with Mad5 and MPLA (equimolar to EDA). Mice werebled 7 days after last vaccination and pentamer staining was performed(one experiment with 3-4 mice per group).

FIG. 9 shows for Example 4 the tumor growth of 7 mice per group(mean±SEM); *, p<0.05 EDAZ13Mad5 versus control group (2-way Anovatest). C57BL/6 mice were implanted s.c. with 3×10⁵ EG7-OVA tumor cellsin the left flank and vaccinated twice (d5 and d13) by subcutaneousinjection of 10 nmol of EDAZ13Mad5, EDAMad5, Mad5 or Mad5 and MPLA(equimolar to EDA) s.c. in the right flank. Tumor size was measured witha caliper.

FIG. 10 shows for Example 4 individual tumor growth curves (7 individualmice per group). C57BL/6 mice were implanted s.c. with 3×10⁵ EG7-OVAtumor cells in the left flank and vaccinated twice (d5 and d13) bysubcutaneous injection of 10 nmol of EDAZ13Mad5, EDAMad5, Mad5 or Mad5and MPLA (equimolar to EDA) s.c. in the right flank. Tumor size wasmeasured with a caliper.

FIG. 11 shows for Example 4 (A) the survival curve of 7 mice per group;*, p<0.05 EDAZ13Mad5 versus control group (Log-rank test) and (B) thetumor-free progression curve of 7 mice per group; *, p<0.05 EDAZ13Mad5versus control group (Log-rank test).

FIG. 12 shows for Example 5 the number of metastasis for everyexperimental group. C57BL/6 mice were implanted i.v. with 1×10⁵ B16-OVAmelanoma tumor cells and vaccinated twice (d0 and d9) by subcutaneousinjection of 2 nmol of EDAZ13Mad5, EDAMad5 or Z13Mad5+MPLA (equimolar toEDA) or MPLA alone s.c. in the right flank. Mice were euthanized at day13 and lung recovered. Number of metastasis foci was counted for eachlung. **, p<0.01; ****, p<0.0001 (Unpaired T test).

FIG. 13 shows for Example 6 the number of metastasis for everyexperimental group. C57BL/6 mice were vaccinated twice (d-21 and d-7) bysubcutaneous injection of 2 nmoles of EDAZ13Mad5, EDAMad5 orZ13Mad5+MPLA (equimolar to EDA) s.c. in the right flank. At day 0, micewere implanted i.v. with 1×10⁵ B16-OVA melanoma tumor cells. Mice wereeuthanized at day 14 and lung recovered. Number of metastasis foci wascounted for each lung. *, p<0.05. ***, p<0.001 (Unpaired T test).

FIG. 14: shows the results for Example 8. HEK-hTLR2 cell lines wereseeded in flat 96-well plate in culture medium, stimulated with 0.3 μM,1 μM or 3 μM of AnaxaZ13Mad5 or Z13Mad5Anaxa and incubated at 37° C. for24 h. Positive control was performed with 500 ng/ml of Pam3CSK4. (A)Twenty microliters of supernatant were added to QuantiBlue® detectionmedium and incubated at 37° C. for 1 h before OD reading (620 nm). (B)Quantification of IL-8 secretion (by ELISA) in the supernatant.

FIG. 15: shows the results for Example 9. C57BL/6 mice were vaccinatedtwice (Wk0 and Wk2) with 2 nmoles of Z13Mad5Anaxa or AnaxaZ13Mad5. Micewere bled 7 days after last vaccination and pentamer staining wasperformed (one experiment).

FIG. 16: shows the results for Example 9. C57BL/6 mice were vaccinatedtwice (Wk0 and Wk2) with 2 nmoles Z13Mad5Anaxa or AnaxaZ13Mad5. Micewere bled 7 days after last vaccination and pentamer staining wasperformed (one experiment with 4 mice per group). *, p<0.05.

FIG. 17: shows for Example 10 the tumor growth of 7 mice per group(mean±SEM). C57BL/6 mice were implanted s.c. with 3×10⁵EG7-OVA tumorcells in the left flank and vaccinated twice (d5 and d13) bysubcutaneous injection of 10 nmol of either AnaxZ13Mad5, Z13Mad5Anaxa orco-injection of Z13Mad5+Pam3CSK4 (equimolar to Anaxa) in the rightflank. Tumor size was measured with a caliper. *, p<0.05; ***, p<0.001,****, p<0.0001.

FIG. 18: shows for Example 10 the individual tumor growth curves (7individual mice per group). C57BL/6 mice were implanted s.c. with 3×10⁵EG7-OVA tumor cells in the left flank and vaccinated twice (d5 and d13)by subcutaneous injection of 10 nmol of either AnaxZ13Mad5, Z13Mad5Anaxaor co-injection of Z13Mad5+Pam3CSK4 (equimolar to Anaxa) s.c. in theright flank. Tumor size was measured with a caliper.

FIG. 19: shows for Example 10 the survival curve of 7 mice per group.C57BL/6 mice were implanted s.c. with 3×10⁵ EG7-OVA tumor cells in theleft flank and vaccinated twice (d5 and d13) by subcutaneous injectionof 10 nmol of either AnaxZ13Mad5, Z13Mad5Anaxa or co-injection ofZ13Mad5+Pam3CSK4 (equimolar to Anaxa) in the right flank. Tumor size wasmeasured with a caliper. *, p<0.05, **, p<0.01, ****, p<0.0001 (Log-ranktest).

FIG. 20: shows for Example 11 the tumor growth of 7 mice per group(mean±SEM). C57BL/6 mice were implanted s.c. with 3×10⁵ EG7-OVA tumorcells in the left flank and vaccinated twice (d5 and d13) bysubcutaneous injection of 2 nmoles of Hp91Z13Mad5, EDAZ13Mad5,Z13Mad5Anaxa, Z13Mad5EDA or Z13Mad5 and MPLA (equimolar to EDA) in theright flank. *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001 (2-wayAnova test at day 23).

FIG. 21: shows for Example 11 the individual tumor growth curves (7individual mice per group). C57BL/6 mice were implanted s.c. with 3×10⁵EG7-OVA tumor cells in the left flank and vaccinated twice (d5 and d13)by subcutaneous injection of 2 nmoles of Hp91Z13Mad5, EDAZ13Mad5,Z13Mad5Anaxa, Z13Mad5EDA or Z13Mad5 and MPLA (equimolar to EDA) s.c. inthe right flank.

FIG. 22: shows for Example 11 the survival curves of all 7 mice pergroup. Median survival is indicated on the graph (m.s.). *, p<0.05; **,p<0.01 (Log-rank test).

FIG. 23: shows for Example 12 the tumor growth of 7 mice per group(mean±SEM); ****, p<0.0001 (Log-rank test). C57BL/6 mice were implanteds.c. with 3×10⁵ EG7-OVA tumor cells in the left flank and vaccinatedtwice (once at d5 and once at d13) by subcutaneous injection of either0.5 nmol, 2 nmol or 10 nmol of Z13Mad5Anaxa in the right flank. Tumorsize was measured with a caliper.

FIG. 24: shows for Example 13 the SIINFEKL-specific CD8 T cell responsesdetected in the blood of C57BL/6 mice vaccinated three times (once atWk0, once at Wk2 and once at Wk4) s.c., i.d. or i.m. with 0.5 nmol (A)or 2 nmol (B) of Z13Mad5Anaxa. Blood was obtained from mice 7 days afterthe 2nd and the 3rd vaccination and multimer staining was performed (oneexperiment with 4 mice per group). *, p<0.05.

FIG. 25: shows for Example 13 KLRG1 expression (A) and PD-1 expression(B), which were analyzed on multimer-positive CD8 T cells (oneexperiment with 4 mice per group). Briefly, C57BL/6 mice were vaccinatedthree times (once at Wk0, once at Wk2 and once at Wk4) s.c., i.d. ori.m. with 2 nmol of Z13Mad5Anaxa. Blood was obtained from mice 7 daysafter the 2nd and the 3rd vaccination and FACS staining was performed.

FIG. 26: shows for Example 14 SIINFEKL-specific CD8 T cell responses inC57BL/6 mice vaccinated two times (once at Wk0 and once at Wk2)intranodally with 0.5 nmol of Z13Mad5Anaxa. Blood was obtained from mice7 days after the 2nd vaccination and multimer staining was performed (3mice per group).

FIG. 27: shows for Example 15 the percentage of pentamer-positive cellsamong CD8 T cells (A and B; *, p<0.05) and KLRG1 geomean ofpentamer-positive CD8 T cells (C and D). Briefly, C57BL/6 mice werevaccinated 3 times (A and C: Wk0, Wk2 and Wk4; B and D: Wk0, Wk2 andWk8) s.c. with 2 nmol of Z13Mad5Anaxa. Mice were bled 7 days after lastvaccination and pentamer staining was performed (one experiment with 4mice per group).

FIG. 28: shows for Example 15 the percentage of multimer-positive cellsamong CD8 T cells (A and D); KLRG1 geomean of multimer-positive CD8 Tcells (B and E) and PD1 geomean of multimer-positive CD8 T cells (C andF). A-C, C57BL/6 mice were vaccinated 3 times at Day0, Day3 and Day7 andbled at Day7 and Day14. D-F, C57BL/6 mice were vaccinated 3 times atDay0, Day7 and Day14 and bled at Day14 and Day21. Vaccination wasperformed s.c. with 0.5 nmol of Z13Mad5Anaxa. Multimer staining wasperformed on blood samples (one

FIG. 29: shows for Example 16 the IL-6 secretion indicating the APCactivation after incubation of BMDCs with various constructs asindicated in the Figure. Briefly, BMDCs were seeded in flat 96-wellplate in culture medium, stimulated with 1 μM of Z13Mad5Anaxa,Mad5Anaxa, Z13Mad5, EDAZ13Mad5 or EDAMad5 and incubated for 24 h at 37°C. IL-6 secretion was quantified by ELISA in the supernatant. Mean±SEMof 2 to 3 individual experiments.

FIG. 30: shows for Example 16 the TNF-α secretion indicating the APCactivation after incubation of Raw 264.7 cells with various constructsas indicated in the Figure. Briefly, Raw 264.7 cells were seeded in flat96-well plate in culture medium, stimulated with 1 μM of Z13Mad5Anaxa,Mad5Anaxa or Z13Mad5 and incubated for 24 h at 37° C. TNF-α secretionwas quantified by ELISA in the supernatant. Mean±SEM of 2 to 3individual experiments.

FIG. 31: shows for Example 17 the IL-8 secretion indicating TLR4 bindingafter incubation of HEK-hTLR4 cells with various constructs as indicatedin the Figure. Briefly, HEK-hTLR4 were seeded in flat 96-well plate inculture medium, stimulated with 1 μM of Z13Mad5Anaxa, Mad5Anaxa,Z13Mad5, EDAZ13Mad5 or EDAMad5 and incubated 24 h at 37° C. IL-8secretion was quantified by ELISA in the supernatant. Mean±SEM of 2individual experiments.

FIG. 32: shows for Example 18 the number of metastasis in a lungmetastasis model with semitherapeutic settings. Briefly, C57BL/6 micewere implanted i.v. with 1×10⁵ B16-OVA melanoma tumor cells andvaccinated twice (d0 and d9) by subcutaneous injection of 2 nmol ofEDAZ13Mad5, Z13Mad5+MPLA (equimolar to EDA) or MPLA alone s.c. in theright flank. Mice were euthanized at day 13 and lung recovered. Numberof metastasis foci was counted for each lung. **, p<0.01 (One-way Anovawith Tukey's multiple comparisons test).

FIG. 33: shows for Example 19 the number of metastasis in a lungmetastasis model with semitherapeutic settings. Briefly, C57BL/6 micewere implanted i.v. with 1×10⁵ B16-OVA melanoma tumor cells andvaccinated twice (d0 and d9) by subcutaneous injection of 0.5 nmol ofZ13Mad5Anaxa, Mad5Anaxa or Z13Mad5+Pam3CSK4 (equimolar to Anaxa) s.c. inthe right flank. Mice were euthanized at day 21 and lung recovered.Number of metastasis foci was counted for each lung. *, p<0.05; **,p<0.01 (Unpaired t-test).

FIG. 34: shows for Example 20 the quantification of SIINFEKL-specificCD8 T cells in a Quad-Gl261 glioblastoma model. Briefly, C57BL/6 micewere implanted i.c. with 5×10⁵ Gl261-Quad tumor cells and vaccinatedtwice (d7 and 21) by s.c. injection of 2 nmol of Z13Mad5Anaxa or 2 nmolof Z13Mad5 and 2 nmol of Anaxa. SIINFEKL-specific CDS T cells werequantified in blood and in BILs at d28 by multimer staining (5-8 miceper group).

FIG. 35: shows for Example 20 the cytokine secretion. Briefly, C57BL/6mice were implanted i.c. with 5×10⁵ Gl261-Quad tumor cells andvaccinated twice (d7 and 21) by s.c. injection of 2 nmol of Z13Mad5Anaxaor 2 nmol of Z13Mad5 and 2 nmol of Anaxa. BILs were isolated andcultured during 6 h with matured BMDCs loaded or not with SIINFEKLpeptide in presence of BrefeldinA before intracellular staining forcytokines. % of CD8 T cells secreting cytokine (5-8 mice per group).

FIG. 36: shows for Example 21 the effect of Z13Mad5Anaxa on survival inthe Quad-GI261 glioblastoma model. Briefly, C57BL/6 mice were implantedi.c. with 5×10⁵ Gl261-Quad tumor cells and vaccinated three times (d7,d21 and d35) by s.c. injection of 2 nmol of Z13Mad5Anaxa. Mice wereweight daily and euthanized when weight loss reached more than 15%.

FIG. 37: shows for Example 22 the effect of Z13Mad5Anaxa on tumor growthand survival in subcutaneous EG7-OVA tumor model in a prophylacticsetting. Briefly, C57BL/6 mice were vaccinated twice (d-21 and d-7) bys.c. injection of 0.5 nmol of Z13Mad5Anaxa in the right flank and thenimplanted at day0 s.c. with 3×10⁵ EG7-OVA tumor cells in the left flank.Tumor size was measured with a caliper. (A) Tumor growth of 7 mice pergroup (mean±SEM); ****, p<0.0001 (2-way Anova test at day 30). (B)Survival curve of 7 mice per group. Median survival is indicated on thegraph (m.s.). ***, p<0.001 (Log-rank test).

FIG. 38: shows for Example 23 the effect of Z13Mad5Anaxa on tumor growthand survival in subcutaneous B16-OVA tumor model in a therapeuticsetting on an established tumor. Briefly, C57BL/6 mice were implanteds.c. with 1×10⁵ B16-OVA tumor cells in the left flank and vaccinatedtwice (d14 and d21) by s.c. injection of 0.5 nmol of Z13Mad5Anaxa in theright flank. (A) Tumor growth of 7 mice per group (mean±SEM); *, p<0.05(2-way Anova test at day 32). (B) Survival curve of 7 mice per group.Median survival is indicated on the graph (m.s.).

FIG. 39: shows for Example 24 the effect of the CPP in Z13Mad5Anaxa ontumor growth and survival in subcutaneous EG7-OVA tumor model. Briefly,C57BL/6 mice were implanted at day0 s.c. with 3×10⁵ EG7-OVA tumor cellsin the left flank and then vaccinated twice (d5 and d13) by s.c.injection of 0.5 nmol of Z13Mad5Anaxa or Mad5Anaxa in the right flank.Tumor size was measured with a caliper. (A) Tumor growth of 7 mice pergroup (mean±SEM); ****, p<0.0001. (B) Survival curve of 7 mice pergroup. Median survival is indicated on the graph (m.s.). **, p<0.01;***, p<0.001.

FIG. 40: shows for Example 25 the effect of complexes having differentCPPs on the immune response. C57BL/6 mice were vaccinated five times(Wk0, Wk2, Wk4, Wk6 and Wk8) s.c. with either 2 nmol (A) or 0.5 nmol (B)of Z13Mad5Anaxa, Z14Mad5Anaxa or Z18Mad5Anaxa. Mice were bled 7 daysafter the 2^(nd), 3^(rd), 4^(th) and 5^(th) vaccination and multimerstaining was performed (one experiment with 4 mice per group). *, p<0.05between vaccinated versus naïve mice at each time point except afterVac2 for Z18Mad5Anaxa-vaccinated mice.

FIG. 41: shows for Example 26 the effect of complexes having differentCPPs on CD8 T cells in spleen (A), draining lymph nodes (B) and bonemarrow (C). C57BL/6 mice were vaccinated five times (Wk0, Wk2, Wk4, Wk6and Wk8) s.c. with 2 nmol of Z13Mad5Anaxa or Z14Mad5Anaxa. Nine daysafter the 5^(th) vaccination, mice were euthanized, organs recovered andmultimer staining was performed.

FIG. 42: shows for Example 26 the effect of complexes having differentCPPs on T cells in spleen (CD8 T cell response (A) and CD4 T cellresponse (B)). C57BL/6 mice were vaccinated five times (Wk0, Wk2, Wk4,Wk6 and Wk8) s.c. with 2 nmol of Z13Mad5Anaxa or Z14Mad5Anaxa. (A) ninedays after the 5^(th) vaccination, Elispot assay was performed on spleencells stimulated with SIINFEKL OVACD8 peptide. (B) nine days after the5^(th) vaccination, Elispot assay was performed on spleen cellsstimulated with OVACD4 peptide.

FIG. 43: shows for Example 26 the effect of complexes having differentCPPs on CD8 T cell effector function. C57BL/6 mice were vaccinated fivetimes (Wk0, Wk2, Wk4, Wk6 and Wk8) s.c. with 2 nmol of Z13Mad5Anaxa orZ14Mad5Anaxa. Nine days after the 5t^(h) vaccination, intracellularstaining was performed on spleen cells stimulated with SIINFEKL OVACD8peptide.

FIG. 44: shows for Example 27 the effect of complexes having differentCPPs on tumor growth (A) and survival rates (B). C57BL/6 mice wereimplanted s.c. with 3×10⁵ EG7-OVA tumor cells in the left flank andvaccinated twice (d5 and d13) by s.c. injection of 0.5 nmol ofZ13Mad5Anaxa or Z14Mad5Anaxa in the right flank. (A) Tumor growth of 7mice per group (mean±SEM); *, p<0.05; ****, p<0.0001 (2-way Anova testat day 28). (B) Survival curve of 7 mice per group. Median survival isindicated on the graph (m.s.). *, p<0.05; **, p<0.01; ***, p<0.001(Log-rank test).

FIG. 45: shows for Example 28 the effect of complexes having differentCPPs on the immune response. C57BL/6 mice were vaccinated three times(Wk0, Wk2 and Wk4) s.c. with 2 nmol (A) or 0.5 nmol (B) of EDAZ13Mad5,EDAZ14Mad5 or EDAZ18Mad5. Mice were bled 7 days after the 3^(rd)vaccination and multimer staining was performed (one experiment with 4mice per group). *, p<0.05

FIG. 46: shows for Example 29 the effect of EDAZ14Mad5 on tumor growth(A) and survival rates (B). C57BL/6 mice were implanted s.c. with 3×10⁵EG7-OVA tumor cells in the left flank and vaccinated twice (d5 and d13)by s.c. injection of 2 nmoles of EDAZ14Mad5 in the right flank. Leftpanel: Tumor growth of 7 mice per group (mean±SEM); **, p<0.01 (2-wayAnova test at day 27). Right panel: Survival curve of 7 mice per group.Median survival is indicated on the graph (m.s.).

FIG. 47: shows for Example 30 the quantification of SIINFEKL-specificCD8 T cells in a Quad-Gl261 glioblastoma model. Briefly, C57BL/6 micewere implanted i.c. with 5×10⁵ Gl261-Quad tumor cells and vaccinatedtwice (d7 and 21) by s.c. injection of 2 nmol of Z13Mad5Anaxa or 2 nmolof Z13Mad5 and 2 nmol of Anaxa. SIINFEKL-specific CD8 T cells werequantified in blood and in BILs at d28 by multimer staining (7-16 miceper group).

FIG. 48: shows for Example 30 the cytokine secretion. Briefly, C57BL/6mice were implanted i.c. with 5×10⁵ Gl261-Quad tumor cells andvaccinated twice (d7 and 21) by s.c. injection of 2 nmol of Z13Mad5Anaxaor 2 nmol of Z13Mad5 and 2 nmol of Anaxa. BILs were isolated andcultured during 6 h with matured BMDCs loaded or not with SIINFEKLpeptide in presence of BrefeldinA before intracellular staining forcytokines. % of CD8 T cells secreting cytokine (7-16 mice per group).

FIG. 49: shows for Example 31 the effect of Z13Mad8Anaxa on T cells inspleen (CD8 T cell response (A) and CD4 T cell response (B)). C57BL/6mice were vaccinated four times (Wk0, Wk2, Wk4 and Wk6) s.c. with 2 nmolof Z13Mad8Anaxa. (A) one week after the 4th vaccination, Elispot assaywas performed on spleen cells stimulated gp70CD8 peptide. (B) one weekafter the 4^(th) vaccination, Elispot assay was performed on spleencells stimulated with gp70CD4 peptide.

FIG. 50: shows for Example 32 the effect of Z13Mad11Anaxa on the numberof metastasis in the B16 lung metastasis model (A) and on the T cellresponse in spleen (B). C57BL/6 mice were vaccinated two times (day0,dayl0) s.c. with 1 nmol of Z13Mad11Anaxa.

FIG. 51: shows for Example 33 the effect of Z13Mad9Anaxa on T cells inspleen (CD8 T cell response. C57BL/6 mice were vaccinated four times(Wk0, Wk2, Wk4 and Wk6) s.c. with 2 nmol of Z13Mad9Anaxa. One week afterthe 4^(th) vaccination, Elispot assay was performed on spleen cellsstimulated with adpgk peptide.

FIG. 52: shows for Example 34 the effect of complexes having differentCPPs on the immune response. C57BL/6 mice were vaccinated two times (Wk0and Wk2) s.c. with 2 nmol of either Z13Mad5Anaxa or TatFMad5Anaxa. Micewere bled 7 days after the 2^(nd) vaccination and multimer staining wasperformed (one experiment with 8 mice per group).

FIG. 53: shows for Example 35 the quantification of SIINFEKL-specificCD8 T cells in naïve mice. Briefly, C57BL/6 mice were vaccinated once(day0) by s.c. injection of 2 nmol of Z13Mad5Anaxa (group“Z13Mad5Anaxa”) or 2 nmol of Z13Mad5 and 2 nmol of Anaxa (group“Z13Mad5+Anaxa”). SIINFEKL-specific CD8 T cells were quantified in bloodat d7 by multimer staining (4-8 mice per group).

FIG. 54: shows for Example 36 the effect of Z13Mad12Anaxa on T cells inblood (CD8 T cell response). C57BL/6 mice were vaccinated twice (Wk0 andWk2) s.c. with 2 nmol of Z13Mad12Anaxa. One week after the 2ndvaccination, multimer staining for the neoantigen reps1 was performed onblood cells.

FIG. 55: shows for Example 37 expression of activation marker H LA-DR,CD83, CD80 and CD86 (from left to right) by human blood monocyte-deriveddendritic cells (DCs) from one single buffy. The DCs were stimulatedwith 300 nM of Z13Mad5Anaxa (lower panels) or Z13Mad5 (upper panels)during 48 h. Isotype staining for each condition was also performed asshown.

EXAMPLES

In the following, particular examples illustrating various embodimentsand aspects of the invention are presented. However, the presentinvention shall not to be limited in scope by the specific embodimentsdescribed herein. The following preparations and examples are given toenable those skilled in the art to more clearly understand and topractice the present invention. The present invention, however, is notlimited in scope by the exemplified embodiments, which are intended asillustrations of single aspects of the invention only, and methods whichare functionally equivalent are within the scope of the invention.Indeed, various modifications of the invention in addition to thosedescribed herein will become readily apparent to those skilled in theart from the foregoing description, accompanying figures and theexamples below. All such modifications fall within the scope of theappended claims.

Example 1 In Vitro Human Dendritic Cell Maturation

The goal of this study was to investigate the capacity of a complexaccording to the present invention to induce maturation of dendriticcells. In the present study, the complex according to the presentinvention is a fusion protein, comprising the cell-penetrating peptide“Z13”, a protein “MAD5”, which consists of different CD8⁺ and CD4⁺epitopes from various antigens, and the TLR4 peptide agonist “EDA”.Accordingly, a fused protein with the EDA peptide at the N-terminalposition and different control conjugated proteins without Z13 or EDA orboth were designed.

Namely, the following constructs were designed, whereby in the aminoacid sequence the cell-penetrating peptide “Z13” is shown underlined andthe TLR peptide agonist “EDA” is shown in italics:

EDAZ13Mad5 Sequence: [SEQ ID NO: 26] MHHHHHHNID RPKGLAFTDV DVDSIKIAWESPQGQVSRYR VTYSSPEDGI RELFPAPDGEDDTAELQGLR PGSEYTVSVV ALHDDMESQP LIGIQSTKRY KNRVASRKSR AKFKQLLQHY REVAAAKSSE NDRLRLLLKE SLKISQAVHA AHAEINEAGREVVGVGALKV PRNQDWLGVP RFAKFASFEA QGALANIAVD KANLDVEQLE SIINFEKLTE WTGS

Molecular weight: 25′057 Da

Characteristics:

-   -   Mad5 cargo contains OVACD4, gp100CD8, EalphaCD4 and OVACD8        epitopes    -   Contains EDA TLR agonist (Lasarte, J. J., et al., The extra        domain A from fibronectin targets antigens to TLR4-expressing        cells and induces cytotoxic T cell responses in vivo. J        Immunol, 2007. 178(2): p. 748-56)    -   Storage buffer: 50 mM Tris-HCl, 150 mM NaCl, 10% Glycerol, 2 mM        DTT, 1 M L-Arginine, pH 8    -   Endotoxin level: <0.01 EU/ug

Z13Mad5 Sequence: [SEQ ID NO: 29]MHHHHHHKRY KNRVASRKSR AKFKQLLQHY REVAAAKSSE NDRLRLLLKE SLKISQAVHAAHAEINEAGR EVVGVGALKV PRNQDWLGVP RFAKFASFEA QGALANIAVD KANLDVEQLESIINFEKLTE WTGS

Molecular weight: 15′196 Da

Characteristics:

-   -   Mad5 cargo contains OVACD4, gp100CD8, EalphaCD4 and OVACD8        epitopes    -   Storage buffer: 50 mM Tris-HCl, 150 mM NaCl, 10% Glycerol, 2 mM        DTT, 1 M L-Arginine, pH 9    -   Endotoxin level:        -   Batch 1: 0.32 EU/mg        -   Batch 2: 0.44 EU/mg

Mad5 Sequence: [SEQ ID NO: 30] MHHHHHHE SLKISQAVHA AHAEINEAGR EVVGVGALKVPRNQDWLGVP RFAKFASFEA QGALANIAVD KANLDVEQLE SIINFEKLTE WTGS

Molecular weight: 10′154.6 Da

Characteristics:

-   -   Mad5 cargo contains OVACD4, gp100CD8, EalphaCD4 and OVACD8        epitopes    -   Storage buffer: 50 mM Tris-HCl, 150 mM NaCl, 10% Glycerol, 2 mM        DTT, 0.5 M L-Arginine, pH 8    -   Endotoxin level: 0.069 EU/mg

The EDAZ13Mad5, Z13Mad5 and Mad5 proteins were investigated for theircapacity to induce human dendritic cell (DC) maturation. Afterincubation during 48 h with 300 nM of protein, activation markersexpression (CD86, CD40, CD83 and HLA-DR) was assessed on the human DCsby FACS (FIGS. 1-4). Specific buffers of each protein were used asnegative controls.

Results are shown for CD40 in FIG. 1, for CD86 in FIG. 2, for HLADR inFIG. 3, and for CD83 in FIG. 4. Whereas EDAZ13Mad5 induced maturation ofhuman DCs, shown by the up-regulation of CD86, HLADR and CD83, Z13Mad5and Mad5 proteins were not able to activate human DCs. These resultsindicate that the EDA portion of the protein is responsible for theup-regulation of the activation markers on the human DCs.

Example 2 In Vitro Epitope Presentation (MHC I)

The goal of this study was to assess functional MHC class I-restrictedcross-presentation in a murine in an vitro system using bone marrowderived dendritic cells (BMDCs) and splenocytes from different TCRtransgenic mice. To this end, the constructs EDAZ13Mad5 and Mad5(described above in Example 1) and the construct EDAMad5 were used:

EDAMad5 Sequence [SEQ ID NO: 31] MHHHHHHNID RPKGLAFTDV DVDSIKIAWESPQGQVSRYR VTYSSPEDGI RELFPAPDGEDDTAELQGLR PGSEYTVSVV ALHDDMESQPLIGIQSTE SLKISQAVHA AHAEINEAGR EVVGVGALKV PRNQDWLGVP RFAKFASFEAQGALANIAVD KANLDVEQLE SIINFEKLTE WTGS

Molecular weight: 20′017 Da

Characteristics:

-   -   Mad5 cargo contains OVACD4, gp100CD8, EalphaCD4 and OVACD8        epitopes    -   Contains EDA TLR agonist    -   Storage buffer: 50 mM Tris-HCl, 150 mM NaCl, 10% Glycerol, 2 mM        DTT, 0.5 M L-Arginine, pH 8    -   Endotoxin level: 1.8 EU/mg

BMDCs were loaded overnight with 300 nM of with the EDAMad5, EDAZ13Mad5and Mad5 proteins containing OVACD8, OVACD4 and gp100 epitopes.Processing and presentation of these MHC I-restricted OVACD8 and gp100epitopes were monitored by measuring the in vitro proliferation of naïveOVA₂₅₇₋₂₆₄-specific CD8⁺ T cells from OT-1 T cell receptor (TCR)transgenic mice and gp100-specific CD8⁺ T cells from P-mel T cell TCRtransgenic mice respectively. Accordingly, efficient MHC classI-restricted presentation of OVACD8 epitope and gp100 epitope wasmonitored after 4 days with CFSE-labeled OT1 cells and P-Mel cellsrespectively. Processing and presentation of MHC Il-restricted OVACD4epitope was monitored by measuring the in vitro proliferation of naïveOVA₃₂₃₋₃₃₉-specific CD4⁺ T cells from OT-2 T cell receptor (TCR)transgenic mice. Accordingly, efficient MHC class II-restrictedpresentation of OVACD4 epitope was monitored after 4 days withCFSE-labeled OT2 cells. As control, BMDCs were pulsed for 1 h with 5 uMpeptide (one experiment representative of 2 individual experiments).

Results are shown in FIG. 5. Similar cross-presentation and processingcapacity of all assessed Mad5-based proteins were observed.

Example 3 CD8 T Cell Immune Response

To investigate the efficacy of EDA-conjugated proteins in inducingpolyclonal CD8⁺ T cell response, C57BL/6 mice were vaccinated twice (Wk0and Wk2), by subcutaneous injection of either 2 nmol or 10 nmol of theconstructs EDAZ13Mad5 or EDAMad5 (described in Examples 1 and 2).Positive control group was vaccinated with Mad5 and the TLR4 agonistMPLA (equimolar to EDA). Two doses were assessed 2 nmol of the construct(FIG. 6) and 10 nmol of the construct (FIG. 7). 3-4 mice were used pergroup.

Seven days after the last vaccination, mice were bled and pentamerstaining was performed to monitor the OVA-specific immune response inthe blood. In FIG. 8, the percentage of pentamer positive CD8+ T cellsis shown for all groups and both doses tested.

These data show that interestingly the immune response is lower at 10nmol compared to 2 nmol. At both doses, 2 nmol and 10 nmol, the vaccinemediated immune response was observed more consistently in theEDAZ13Mad5 group in contrast to the EDAMad5 group. Moreover, there is anincreased immune response when the TLR4 agonist is conjugated with thevaccine.

Example 4 Vaccine Efficacy on Tumor Growth in a Benchmark EG.7-OVA TumorModel

To evaluate the effect of EDA construct proteins on tumor growthcontrol, the s.c. model of EG.7-OVA thymoma cells was chosen. C57BL/6mice were implanted s.c. with 3×10⁵ EG7-OVA tumor cells in the leftflank. After tumor implantation, mice were vaccinated at day 5 and 13with 10 nmol of one of the following constructs (cf. Examples 1 and 2for construct description): EDAZ13Mad5, EDAMad5, Mad5, or Mad5 and MPLA(equimolar to EDA) s.c. in the right flank. Tumor size was measured witha caliper.

FIG. 9 shows the tumor growth of 7 mice per group (mean±SEM); *, p<0.05EDAZ13Mad5 versus control group (2-way Anova test). FIG. 10 showsindividual tumor growth curves (7 individual mice per group). FIG. 11Ashows the survival curve of 7 mice per group; *, p<0.05 EDAZ13Mad5versus control group (Log-rank test). FIG. 11B shows the tumor-freeprogression curve of 7 mice per group; *, p<0.05 EDAZ13Mad5 versuscontrol group (Log-rank test).

The results show that in a therapeutic setting, EDAZ13Mad5 was the onlyprotein vaccine to significantly control the tumor growth compared tothe control group with a significant better tumor free progression curveand survival curve.

The results therefore suggest that the construct protein EDAZ13Mad5 is ahighly potent vaccine for controlling the tumor growth in a therapeuticsetting.

Example 5 Vaccine Efficacy on Tumor Growth in a Melanoma MetastasisModel

To assess the efficacy in a lung metastasis model using B16-OVA tumorcells in a semi-therapeutic setting, different construct proteins wereused: EDAMad5, EDAZ13Mad5, Z13Mad5+MPLA (cf. Examples 1 and 2 for designof the constructs), and MPLA alone. C57BL/6 mice were implanted i.v.with 1×10⁵ B16-OVA melanoma tumor cells and at the same time (d0) 2 nmolof the vaccine (EDAMad5, EDAZ13Mad5, Z13Mad5+MPLA, MPLA alone) wasadministered by subcutaneous injection in the right flank. Nine dayslater, mice were vaccinated a second time with the same dose. Furthercontrol groups were vaccinated with 2 nmol of Z13Mad5 and the TLR4agonist MPLA (equimolar to EDA) or MPLA alone. Mice were euthanized atday 13 and lung recovered. Number of metastasis foci was counted foreach lung. The results are shown in FIG. 12.

The results show that the conjugate EDAZ13Mad5 is as potent asZ13Mad5+MPLA to inhibit tumor metastasis in the lung. Furthermore,EDA-Mad5 is less potent than EDAZ13Mad5, indicating a crucial role ofZ13 in vaccine efficacy.

Example 6 Vaccine Efficacy on Tumor Growth in a Melanoma MetastasisModel—Prophylactic Setting

Furthermore, the efficacy of the different construct proteins EDAMad5,EDAZ13Mad5, and Z13Mad5+MPLA (cf. Examples 1 and 2 for design of theconstructs) was assessed in a lung metastasis model in a prophylacticsetting. C57BL/6 mice were vaccinated 21 and 7 days before implantationof tumor cells (d-21 and d-7) by subcutaneous injection of 2 nmol ofEDAZ13Mad5, EDAMad5 or Z13Mad5+MPLA (equimolar to EDA) s.c. in the rightflank. At day 0, mice were implanted i.v. with 1×10⁵ B16-OVA melanomatumor cells. Mice were euthanized at day 14 and lung recovered. Resultsare shown in FIG. 13.

Example 7 Design of further Constructs comprising a TLR2 Peptide Agonist

Herein, the complex according to the present invention is again a fusionprotein, comprising the cell-penetrating peptide “Z13”, the protein“MAD5”, which consists of different CD8⁺ and CD4⁺ epitopes from variousantigens, and the TLR2 peptide agonist “Anaxa”. Accordingly, fusedproteins with the Anaxa peptide at the C-terminal or N-terminal positionwere designed.

Namely, the following constructs were designed, whereby in the aminoacid sequence the cell-penetrating peptide “Z13” is shown underlined andthe TLR peptide agonist “Anaxa” is shown in italics:

AnaxaZ13Mad5 Sequence: [SEQ ID NO: 27] MHHHHHHSTV HEILCKLSLE GDHSTPPSAYGSVKPYTNFD AE KRYKNRVA SRKSRAKFKQ LLQHYREVAA AKSSENDRLR LLLKESLKISQAVHAAHAEI NEAGREVVGV GALKVPRNQD WLGVPRFAKF ASFEAQGALA NIAVDKANLDVEQLESIINF EKLTEWTGS

Molecular weight: 18973 Da

Characteristics:

-   -   Mad5 cargo contains OVACD4, gp100CD8, EalphaCD4 and OVACD8        epitopes    -   Contains the 35-mer peptide of Annexin    -   Storage buffer: 50 mM Tris-HCl, 150 mM NaCl, 10% Glycerol, 2 mM        DTT, 0.5 M L-Arginine, pH 8    -   Endotoxin level: 5.17 EU/mg

Z13Mad5Anaxa Sequence: [SEQ ID NO: 28]MHHHHHHKRYKNRVA SRKSRAKFKQ LLQHYREVAA AKSSENDRLR LLLKESLKIS QAVHAAHAEINEAGREVVGV GALKVPRNQD WLGVPRFAKF ASFEAQGALA NIAVDKANLD VEQLESIINFEKLTEWTGSS TVHEILCKLS LEGDHSTPPS AYGSVKPYTN FDAE

Molecular weight: 18973 Da

Characteristics:

-   -   Mad5 cargo contains OVACD4, gp100CD8, EalphaCD4 and OVACD8        epitopes    -   Contains the 35-mer peptide of Annexin    -   Storage buffer: 50 mM Tris-HCl, 150 mM NaCl, 10% Glycerol, 2 mM        DTT, 0.5 M L-Arginine, pH 8

Endotoxin level: 3.1 EU/mg

Example 8 TLR2 Binding (HEK-hTLR2 Cell Lines)

The goal of this study was to assess whether the Z13Mad5Anaxa andAnaxaZ13Mad5 construct proteins (cf. Example 7 for design of theseconstruct proteins) were able to bind TLR2 as an agonist. HEK-Blue™hTLR2 were seeded in flat 96-well plate in culture medium, stimulatedwith 0.3 μM, 1 μM or 3 μM of AnaxaZ13Mad5 or Z13Mad5Anaxa and incubatedat 37° C. for 24 h. Positive control was performed with 500 ng/ml ofPam3CSK4, a TLR2 agonist.

To monitor the activation of NF-κB/AP1, twenty microliters of thesupernatant were added to QuantiBlue® detection medium and incubated at37° C. for 1 h before OD reading (620 nm). Results are shown in FIG.14A.

The secretion of IL-8 in the supernatant was quantified by ELISA.Results are shown in FIG. 14B.

Results (FIG. 14A, B) showed that Z13Mad5Anaxa and AnaxaZ13Mad5 aresimilarly able to bind to TLR2 in a dose dependent manner.

Example 9 In Vivo Induction of Specific CD8⁺ T Cells

To investigate the efficacy of the Anaxa-conjugated proteins of Example7 in the induction of CD8⁺ T cell responses, C57BL/6 mice werevaccinated twice (Wk0 and Wk2), by subcutaneous injection of 2 nmol ofAnaxaZ13Mad5 or 2 nmol of Z13Mad5Anaxa. Seven days after the lastvaccination, mice were bled and to monitor the OVA-specific immuneresponse in the blood, pentamer staining was performed (one experimentwith 4 mice per group). Results are shown in FIGS. 15 and 16.

These data indicate that both, the Z13Mad5Anaxa vaccine and theAnaxaZ13Mad5 construct, elicit a strong immune response.

Example 10 Therapeutic Effect on Tumor Growth

To evaluate the effect of the Anaxa-conjugated construct proteinsdesigned in Example 7 on tumor growth control, a benchmark tumor modelwas used, namely the s.c. implantation of EG.7-OVA thymoma cells.

C57BL/6 mice were implanted s.c. with 3×10⁵ EG7-OVA tumor cells in theleft flank. After tumor implantation, the three groups of 7 mice eachwere vaccinated s.c. in the right flank at day 5 and 13 by subcutaneousinjection of 10 nmol of either AnaxZ13Mad5 (group 1), Z13Mad5Anaxa(group 2) or Z13Mad5 and Pam3CSK4 (equimolar to Anaxa; group 3). Inorder to compare the effect to a protein mixed with an externaladjuvant, a control group was vaccinated with Z13Mad5 and Pam3CSK4(equimolar to Anaxa). Tumor size was measured with a caliper. Resultsare shown in FIG. 17-19.

In a therapeutic schedule, Z13Mad5Anaxa and AnaxaZ13Mad5 are betterprotein vaccines for controlling tumor growth compared to the controlgroup, i.e. co-injection of Z13Mad5 and Pam3CSK showing a significantbetter survival curve. In particular, Z13Mad5Anaxa and AnaxaZ13Mad5demonstrate significantly higher efficacy than Z13Mad5 administratedseparately with Pam3CSK4. The results therefore suggest that theconstruct proteins Z13Mad5Anaxa and AnaxaZ13Mad5 are promisingconjugate-vaccines for controlling the tumor growth in a therapeuticsetting.

Example 11 Therapeutic Effect on Tumor Growth—Comparison of Constructswith Different TLR Agonists

The goal of this study was to compare the efficacy of the differentconstruct protein vaccines conjugated to different TLR agonist, namelyEDAZ13Mad5 and Z13Mad5Anaxa of Example 1 and 7, on tumor growth control.To this end, C57BL/6 mice were implanted s.c. with 3×10⁵ EG.7-OVAthymoma cells in the left flank as described previously in Example 10.Mice (7 individual mice per group) were vaccinated s.c. in the rightflank at day 5 and 13 with 2 nmol of either EDAZ13Mad5, Z13Mad5Anaxa orco-injection of Z13Mad5+MPLA (equimolar to EDA).

Results are shown in FIGS. 20, 21 and 22. In this experimental setting,Z13Mad5Anaxa, EDAZ13Mad5, and Z13Mad5+MPLA were similarly able tosignificantly control tumor growth. Moreover, these data indicate thatZ13Mad5Anaxa is the best construct to significantly control tumor growthand EDAZ13Mad5 was slightly better than Z13Mad5+MPLA in thisexperimental setting.

Example 12 Dose Effect of Z13Mad5Anaxa on Tumor Growth Control

To identify the optimal dose of the conjugate vaccine, three differentdoses (0.5 nmol, 2 nmol and 10 nmol) of Z13Mad5Anaxa (cf. Example 7)were assessed for their ability to control tumor growth. The dose effectof Z13Mad5Anaxa construct was evaluated in the s.c. model of EG.7-OVAthymoma cells as described previously in Example 10. After tumorimplantation, mice were vaccinated twice (at day 5 and at day 13 aftertumor implantation) in a therapeutic setting at 0.5, 2 or 10 nmol ofZ13Mad5Anaxa.

C57BL/6 mice were implanted s.c. with 3×10⁵ EG7-OVA tumor cells in theleft flank and vaccinated twice (d5 and d13) by subcutaneous injectionof either 0.5 nmol, 2 nmol or 10 nmol of Z13Mad5Anaxa in the rightflank. Tumor size was measured with a caliper.

The tumor growth of 7 mice per group is depicted in FIG. 23. Those datashow that the doses of 0.5 and 2 nmol are at least as efficacious as 10nmol for controlling tumor growth.

Example 13 Effect of Different Routes of Administration of Z13Mad5Anaxa

This study was based on the previous Examples demonstrating the efficacyof Z13Mad5Anaxa conjugate vaccine (cf. Example 7), which is able toelicit specific immune responses and is efficacious for controllingtumor growth in the subcutaneous tumor model EG7 as shown above.

To investigate the effect of subcutaneous, intramuscular and intradermalroutes of administration, immune responses elicited by subcutaneous,intramuscular and intradermal injection were compared. Intradermalinjections were performed using the PLEASE® device from PantecBiosolutions.

Mice were vaccinated three times every two weeks (Wk0, Wk2 and Wk4) with0.5 or 2 nmol of Z13Mad5Anaxa (cf. Example 7). In order to targetseveral lymph nodes, the 1st and the 3rd vaccinations were performed inthe right flank whereas the 2nd was done in the left flank.SIINFEKL-specific CD8+ T cell response was analyzed 1 week after the 2ndand the 3rd vaccination in the blood. FIG. 24 shows theSIINFEKL-specific CD8 T cell responses after each vaccination detectedin the blood of C57BL/6 mice vaccinated three times (Wk0, Wk2 and Wk4)s.c., i.d. or i.m. with 0.5 nmol (FIG. 24A) or 2 nmol (FIG. 24B) ofZ13Mad5Anaxa. Blood was obtained from mice 7 days after the 2nd and the3rd vaccination and multimer staining was performed (one experiment with4 mice per group).

The results indicate that at the two doses assessed (0.5 and 2 nmol),(i) all routes of administration tested elicited a SIINFEKL-specific CD8immune response and (ii) the subcutaneous vaccination elicited thestrongest SIINFEKL-specific CD8 immune response. For subcutaneousadministration, the maximum response was reached after the 3ndvaccination and still maintained after the 3rd vaccination. TheSIINFEKL-specific CD8 immune response after the 2nd vaccination elicitedby intradermal and intramuscular vaccinations is lower compared tosubcutaneous vaccination and is not enhanced after the 3rd vaccination.

Next, the effector function and the exhaustion status ofSIINFEKL-specific CD8 T cells was evaluated by analyzing KLRG 1 (Killercell lectin-like receptor subfamily G member 1) and PD-1 respectively.

To this end, C57BL/6 mice were vaccinated three times (Wk0, Wk2 and Wk4)s.c., i.d. or i.m. with 2 nmol of Z13Mad5Anaxa (cf. Example 7). Bloodwas obtained from mice 7 days after the 2nd and the 3rd vaccination andFACS staining was performed. KLRG1 and PD-1 expression were analyzed onmultimer-positive CD8 T cells (one experiment with 4 mice per group).Results are shown in FIG. 25.

These data indicate that the expression of KLRG 1 is strongly increasingon SIINFEKL-specific CD8 T cells after subcutaneous vaccination. Afteri.d. or i.m. vaccination, the observed effects were lower. Thepercentage of KLRG 1-positive cells among SIINFEKL-specific CD8 T cellsis also enhanced after s.c. vaccination (data not shown).

In contrast to KLRG 1, PD-1 expression is decreasing with the time andthe vaccinations, for subcutaneous and intramuscular vaccination routes.This suggests that SIINFEKL-specific CD8 T cells are not exhausted. Thepercentage of PD1-positive cells among SIINFEKL-specific CD8 T cells isalso reduced after s.c. and i.m. vaccination (data not shown). It isimportant to note that PD-1 expression is higher after the 2ndvaccination when mice were vaccinated subcutaneously, reflecting theearly activation status of specific T cells (Keir, M. E., et al., PD-1and its ligands in tolerance and immunity. Annu Rev Immunol, 2008. 26:p. 677-704).

The expression of the late exhaustion marker Tim-3 was also analyzed. Avery low expression as observed for all groups.

Taken together, results indicate that subcutaneous vaccination elicitsthe best specific CD8 immune response compared to intramuscular orintradermal injections.

Example 14 Intranodal Route of Administration

Based on the previous experiments (Example 13), the intranodal route ofadministration was additionally investigated. To this end, the immuneresponse elicited by intranodal injection of Z13Mad5Anaxa (cf. Example7) was investigated.

For this purpose, mice were first injected with Evans Bluesubcutaneously in order to allow easily visualizing the lymph nodes forinjection and inject intranodally without invasive surgery, for exampleas described in Jewell, C. M., S. C. Lopez, and D. J. Irvine, In situengineering of the lymph node microenvironment via intranodal injectionof adjuvant-re/easing polymer particles. Proc Natl Acad Sci USA, 2011.108(38): p. 15745-50.

C57BL/6 mice were vaccinated two times every two weeks (Wk0 and Wk2)intranodally with 0.5 nmol of Z13Mad5Anaxa (cf. Example 7). The 1stvaccination was performed in the right inguinal lymph node, whereas thesecond vaccination was done in the left inguinal lymph node. Blood wasobtained from mice 7 days after the 2nd vaccination and multimerstaining was performed (3 mice per group). In other words,SIINFEKL-specific CD8+ T cell response was analyzed one week after the2nd vaccination in the blood. FIG. 26 shows the SIINFEKL-specific CD8 Tcell responses. Those data indicate that also intranodal injection wasable to elicit SIINFEKL-specific CD8 T cells.

Example 15 Vaccination Schedule

The vaccination schedule evaluation work was initiated with theobjective to identify the impact of the third vaccination using the sameZ13Mad5Anaxa construct as described above (cf. Example 7). Thesubcutaneous route was chosen given the previous results.

In the experiment first two vaccinations were performed at wk0 and wk2with a 3rd vaccination either at wk4 (FIG. 27A) or at wk8 (FIG. 27B).Thus, C57BL/6 mice were vaccinated three times (FIG. 27A and C: Wk0, Wk2and Wk4 and FIG. 27B and D: Wk0, Wk2 and Wk8) s.c. with 2 nmol ofZ13Mad5Anaxa. Blood was obtained from mice 7 days after last vaccinationand pentamer staining was performed (one experiment with 4 mice pergroup). Accordingly, SIINFEKL-specific CD8+ T cell response was analyzed1 week after the 2nd and the 3rd vaccination (FIGS. 27A and B).Additionally, the effector function of SIINFEKL-specific T cells wasevaluated by analyzing the expression of KLRG 1 on specific CD8 T cells(FIGS. 27C and D).

The data indicate that compared to control the percentage ofSIINFEKL-specific CD8 T cells was significantly increased at all timepoints tested (Vac2 and Vac3) as well as in both vaccination schedules(FIGS. 27A and B).

Interestingly, the third vaccination at Wk4 allowed to most prominentlyincreasing the percentage of SIINFEKL-specific CD8 T cells (FIG. 27A).The same cells also demonstrate an improved effector function throughhigher KLRG 1 expression (FIG. 27C). In contrast, with a thirdvaccination performed at Wk8 no improvement from the second to the thirdvaccination could be observed in the SIINFEKL-specific immune responseand in the KLRG 1 expression.

Taken together, these results indicate that the CD8 immune responsecould be increased by shorten the delay between the second and the thirdvaccination.

Given that an earlier third vaccination seems to increase immuneresponse, in the next study two short schedules of vaccination wereinvestigated:

i) three vaccinations at day 0, day 3 and day 7 and

ii) three vaccinations at day 0, day 7 and day 14.

Again, C57BL/6 mice were used and vaccination was performed s.c. with0.5 nmol of Z13Mad5Anaxa (cf. Example 7). Multimer staining wasperformed on blood samples obtained one week after the 2nd and the 3rdvaccination (one experiment with 4 mice per group).

Thus, SIINFEKL-specific CD8+ T cell response was analyzed one week afterthe 2nd and the 3rd vaccination (FIGS. 28A and D). Additionally, theeffector function of SIINFEKL-specific T cells was evaluated byanalyzing the expression of KLRG 1 on specific CD8 T cells (FIGS. 28Band 28E) and the exhaustion status by analyzing the PD-1 expression ofspecific T cells (FIGS. 28C and 28F).

The data indicate that—similarly to the first study regarding thevaccination schedule described above—compared to control the percentageof SIINFEKL-specific CD8 T cells was increased at all time points tested(Vac2 and Vac3) as well as in both vaccination schedules (FIGS. 28A andB).

However, compared to the schedule wk0-wk2-wk4, a schedule withvaccinations at Day0, Day3 and Day7 did not elicit such a highSIINFEKL-specific CD8 T cell immune response. Concerning the schedulewith vaccinations at Day0, Day7 and Dayl4, the SIINFEKL-specific CD8 Tcell immune response elicited is better compared to the previousschedule (d0-d3-d7) but is not maintained after the 3rd vaccination.

Taken together, vaccination schedule data set indicates that theWk0-Wk2-Wk4 vaccination schedule is the best vaccination schedule forinducing potent OVA-specific CD8 immune response with high effectorfunction.

Example 16 Capacity of TLR Agonist-CPP Conjugate Constructs to ActivateMurine Antigen-Presenting Cells (APCs)

To investigate the effect of both, the CPP component and the TLR agonistcomponent in a complex according to the present invention, again thefusion proteins as described above (cf. Examples 1, 2 and 7) were used.

In addition, a further “control peptide” was designed, which is also afusion protein and which comprises the protein “MAD5”, which consists ofdifferent CD8⁺ and CD4⁺ epitopes from various antigens, and the TLR2peptide agonist “Anaxa” (i.e. without cell penetrating peptide).Accordingly, the following control construct was additionally designed:

Mad5Anaxa Sequence: [SEQ ID NO: 32] MHHHHHHESL KISQAVHAAH AEINEAGREVVGVGALKVPR NQDWLGVPRF AKFASFEAQG ALANIAVDKA NLDVEQLESI INFEKLTEWTGSSTVHEILC KLSLEGDHST PPSAYGSVKP YTNFDAE

Molecular weight: 13933 Da

Characteristics:

-   -   Mad5 cargo contains OVACD4, gp100CD8, EalphaCD4 and OVACD8        epitopes    -   Contains the 35-mer peptide of Annexin in C-terminal position    -   Storage buffer: 50 mM Tris-HCl, 150 mM NaCl, 10% Glycerol, 2 mM        DTT, 0.5 M L-Arginine, pH 8    -   Endotoxin level: Batch 1—12.15 EU/mg

The aim of this study was to evaluate the capacity of two exemplarycomplexes according to the present invention, namely EDAZ13Mad5 (cf.Example 1) and Z13Mad5Anaxa (cf. Example 7), to promoteantigen-presenting cells activation in comparison to reference complexeslacking either the cell penetrating peptide component Z13 (Mad5Anaxa,cf. above; EDAMad5, cf. Example 2) or the TLR agonist (Z13Mad5, cf.Example 1).

To this end, the capacity of the above mentioned constructs to promoteantigen-presenting cells (APC) activation was assessed in bonemarrow-derived dendritic cells (BMDCs), which express all TLRs exceptTLR7.

BMDCs were seeded in flat 96-well plate in culture medium, stimulatedwith 1 μM of either Z13Mad5Anaxa (cf. Example 7), Mad5Anaxa (cf. above),Z13Mad5 (cf. Example 1), EDAZ13Mad5 (cf. Example 1) or EDAMad5 (cf.Example 2) and incubated for 24 h at 37° C.

The APC activation was investigated by monitoring the secretion of IL-6in the culture supernatant of BMDCs. IL-6 secretion was quantified byELISA in the supernatant.

The results are shown in FIG. 29. These data clearly show thatZ13Mad5Anaxa was able to activate BMDCs, whereas no such activation wasobserved when the cells were cultured in presence of Z13Mad5 orMad5Anaxa. This suggests that not only the TLR agonist (Anaxa or EDA) iscritical for the activation of macrophages and dendritic cells, but thatthe CPP is also needed. Also the presence of the CPP without the TLRagonist is not sufficient, but indeed both, CPP and TLR agonist arecritical for the activation of macrophages and dendritic cells.

Those results were confirmed by using another cell line, namely in theRaw 264.7 mouse macrophage cell line, which expresses all TLRs exceptTLR5 (Applequist, S. E., R. P. Wallin, and H. G. Ljunggren, Variableexpression of Toll-like receptor in murine innate and adaptive immunecell lines. Int Immunol, 2002. 14(9): p. 1065-74).

Raw 264.7 cells were seeded in flat 96-well plate in culture medium,stimulated with 1 μM of either Z13Mad5Anaxa (cf. Example 7), Mad5Anaxa(cf. above) or Z13Mad5 (cf. Example 1) and incubated for 24 h at 37° C.

In Raw 264.7 cells the APC activation was investigated by monitoring thesecretion of TNF-α in the culture supernatant of Raw 264.7. TNF-asecretion was quantified by ELISA in the supernatant. The results areshown in FIG. 30.

It is thought that the CPP may facilitate the entry of the molecule intothe cells, allowing a better targeting of intracellular TLR.

Taken together, the data reveal the critical role of both, CPP and TLRagonist, within the conjugate constructs to activate APC. This effectmay be due to helping the entry of the construct into the cells,therefore resulting in an optimal targeting of the intracellular TLR.

Example 17 Ability of the Conjugate Constructs to Bind to Human TLR4

It was recently shown that the Anaxa peptide owns an adjuvant activityby signaling through TLR2 (WO 2012/048190 A1), whereas the EDA peptideis a natural ligand for TLR4 (Okamura, Y., et al., The extra domain A offibronectin activates Toll-like receptor 4. J Biol Chem, 2001. 276(13):p. 10229-33).

Moreover, as shown above in Example 8 and FIG. 14, a complex accordingto the present invention comprising the Anaxa peptide as TLR agonist,for example Z13Mad5Anaxa, is able to bind to human TLR2 and to promotethe secretion of IL-8 by HEK-hTLR2 cells (cf. Example 8, FIG. 14).

In the present study, the ability of complexes according to the presentinvention comprising either the Anaxa peptide as TLR agonist or the EDApeptide as TLR agonist to bind to human TLR4 was evaluated. To this end,HEK cells transfected with human TLR4 (HEK-hTLR4) were seeded in flat96-well plate in culture medium, stimulated with 1 μM of eitherZ13Mad5Anaxa (cf. Example 7), Mad5Anaxa (cf. above), Z13Mad5 (cf.Example 1), EDAZ13Mad5 (cf. Example 1) or EDAMad5 (cf. Example 2) andincubated for 24 h at 37° C. IL-8 secretion was quantified by ELISA inthe supernatant.

Results are shown in FIG. 31. As expected, incubation of HEK-hTLR4 withEDAZ13Mad5 resulted in remarkable IL-8 secretion, indicating binding ofEDAZ13Mad5 to TLR4. In line with the results obtained in Example 16, theIL-8 secretion of EDAMad5 (without the CPP) was remarkably lower ascompared to EDAZ13Mad5, showing the effect of the presence of a CPP. TheZ13Mad5 construct, which does not comprise a TLR agonist, showed no IL-8secretion, indicating—as expected—a lack of binding to TLR4.

Interestingly, incubation of HEK-hTLR4 with the construct Z13Mad5Anaxaresulted in the most pronounced IL-8 secretion, indicating binding ofZ13Mad5Anaxa to TLR4. This is astonishing, since Anaxa was previouslyhypothesized to be a TLR2 agonist. Again, the same construct but withoutthe CPP (Mad5Anaxa) resulted in remarkably lower IL-8 secretion,confirming the results obtained in Example 16.

Taken together, these data (i) confirm the results obtained in Example16, (ii) confirm that EDA is indeed a TLR4 agonist, and (iii) showsurprisingly that the Anaxa peptide is also a TLR4 agonist (in additionto being a TLR2 agonist, cf. Example 8 and FIG. 14).

Example 18 Vaccine Efficacy on Tumor Growth in a Lung MetastasisModel—Semi-Therapeutic Setting: TLR Agonist EDA

This study is based on Example 6, showing the efficacy of a complexaccording to the present invention, namely EDAZ13Mad5, in a melanomalung metastasis model in a prophylactic setting (cf. FIG. 13).

In the present study the same lung metastasis model was used as well asthe construct proteins EDAZ13Mad5 and Z13Mad5+MPLA (cf. Examples 1 and 2for design of the constructs). However, in the semi-therapeutic setting,C57BL/6 mice were vaccinated at the same time as tumor cells wereimplanted (d0) and, for a second time, at nine days after implantation(d9). Vaccination was performed by subcutaneous injection of 2 nmol ofEDAZ13Mad5, Z13Mad5+MPLA (equimolar to EDA) or MPLA s.c. in the rightflank. At day 0, mice were implanted i.v. with 1×10⁵ B16-OVA melanomatumor cells and vaccinated twice (d0 and d9) by subcutaneous injectionof 2 nmol of EDAZ13Mad5, Z13Mad5+MPLA (equimolar to EDA) or MPLA alones.c. in the right flank. Mice were euthanized at day 13 and lungrecovered.

Results are shown in FIG. 32.

The results show that EDAZ13Mad5 is slightly more potent thanZ13Mad5+MPLA to inhibit the growth of melanoma metastasis. In addition,no adjuvant effect was observed in mice injected with MPLA only.

Both, EDAZ13Mad5 and Z13Mad5+MPLA, significantly inhibit the growth ofmelanoma metastasis in the lung in prophylactic and semitherapeuticsettings.

Example 19 Vaccine Efficacy on Tumor Growth in a Lung MetastasisModel—Semi-Therapeutic Setting: TLR Agonist Anaxa

This study is based on Example 18 with the same model (semitherapeuticsettings) and experimental schedule. However, the effect of complexesaccording to the present invention comprising the “Anaxa” peptide as TLRagonist were investigated—instead of the EDA TLR agonist as in Example18.

To this end, C57BL/6 mice were implanted i.v. with 1×10⁵ B16-OVAmelanoma tumor cells and vaccinated twice (d0 and d9) by subcutaneousinjection of 0.5 nmol of Z13Mad5Anaxa, Mad5Anaxa or Z13Mad5+Pam3CSK4(equimolar to Anaxa) s.c. in the right flank. Mice were euthanized atday 21 and the lung was recovered. Number of metastasis foci was countedfor each lung. The results are shown in FIG. 33.

The results show that Z13Mad5Anaxa is sensibly more potent thanZ13Mad5+Pam3CSK4 to inhibit the growth of melanoma metastasis. Incontrast, Mad5Anaxa was not able to control metastasis growth in thelung, underlining again the importance of CPP.

Altogether, the B16-OVA lung metastasis experiment showed thatZ13Mad5Anaxa was highly efficacious in inhibiting the growth of melanomametastasis in the lung.

Example 20 Vaccine Efficacy in a Glioblastoma Model

In this study, another cancer model was used, namely a glioblastomamodel. Glioma is the most frequent form of primary brain tumors inadults, with glioblastoma multiforme (GBM) being the most lethal. Thistumor is notorious for its highly invasive and aggressive behavior.Currently, the best treatment against GBM is a regimen involving acombination of surgery, chemotherapy and radiotherapy, which has amedian survival period of only 14.6 months. There is an urgent, unmetmedical need for new treatment modalities that improve the prognosis ofglioma patients. T-cell mediated immunotherapy is a conceptuallyattractive treatment option to use in conjunction with existingmodalities for glioma, in particular highly invasive GBM.

The Gl261 glioma is a carcinogen-induced mouse glioma model. This modelrepresents one of the very few brain tumor models developed inimmunocompetent animals, that has growth characteristics similar tohuman GBM (Newcomb, E. and D. Zagzag, The murine GL261 gliomaexperimental model to assess novel brain tumor treatments, in CNS CancerModels, Markers, Prognostic, Factors, Targets, and TherapeuticApproaches, E. G. Van Meir, Editor. 2009, Humana Press: Atlanta. p.227-241; Jacobs, V. L., et al., Current review of in vivo GBM rodentmodels: emphasis on the CNS-1 tumour model. ASN Neuro, 2011. 3(3): p.e00063). Low numbers of intracranially transplanted Gl261 cells formedintracranial tumors in C57BL/6 mice (Zhu, X., et al., Poly-/CLC promotesthe infiltration of effector T cells into intracranial gliomas viainduction of CXCL 10 in IFN-alpha and IFN-gamma dependent manners.Cancer Immunol lmmunother, 2010. 59(9): p. 1401-9; Zhu, X., et al., Tolllike receptor-3 ligand poly-/CLC promotes the efficacy of peripheralvaccinations with tumor antigen-derived peptide epitopes in murine CNStumor models. J Transl Med, 2007. 5: p. 10). The cells are moderatelyimmunogenic: they are able to elicit tumor-specific immune response atthe tumor site. However, the tumor-specific immune cells are not capableof complete tumor clearance.

Recently, M. Ollin generated a new Gl261 model (Ohlfest, J. R., et al.,Vaccine injection site matters: qualitative and quantitative defects inCDB T cells primed as a function of proximity to the tumor in a murineglioma model. J Immunol, 2013. 190(2): p. 613-20) by transfecting Gl261cell line with the “Quad Cassette” expressing four peptides presented byH-2b class I or II molecules: human gp100₂₅₋₃₃, chicken OVA₂₅₇₋₂₆₄,chicken OVA₃₂₃₋₃₃₉, and mouse I-Eα₅₂₋₆₈. The Quad-Gl261 cell line alsostably expresses luciferase, which allows the follow-up of tumor growthby bioluminescence.

The goal of this study was to assess the efficacy of a complex accordingto the present invention in the Quad-Gl261 glioblastoma model.

The effect of a complex according to the present invention, namelyZ13Mad5Anaxa (cf. Example 7) was evaluated in the above describedglioblastoma model. T cell homing at the tumor site was thereforeanalyzed in Gl261-Quad tumor-bearing mice vaccinated twice (Wk1 and Wk3)with Z13Mad5Anaxa vaccine. A group vaccinated with Z13Mad5 and Anaxa(equimolar to Z13Mad5Anaxa) administrated separately was used ascontrol. Briefly, C57BL/6 mice were implanted i.c. (intracranially) with5×10⁵ Gl261-Quad tumor cells and vaccinated twice (at d7 and d21following implantation) by s.c. injection of 2 nmol of Z13Mad5Anaxa(group 1) or 2 nmol of Z13Mad5 and 2 nmol of Anaxa (group 2). At Wk4,the blood and the brain infiltrating leukocytes (BILs) were analyzed,whereby SIINFEKL-specific CD8 T cells were quantified in blood and inBILs at d28 by multimer staining (5-8 mice per group). Results are shownin FIG. 34.

In general, low frequency of SIINFEKL-specific CD8 T cells wasquantified in the blood. However, a higher percentage ofSIINFEKL-specific CD8 T cells was observed in the blood ofZ13Mad5Anaxa-vaccinated mice. In all groups, there was a sensiblystronger accumulation of SIINFEKL-specific CD8 T cells in the BILs.

After two vaccinations with Z13Mad5Anaxa, the frequency ofSIINFEKL-specific cells CD8+ T cells in the BILs was 2-fold higher (24%)than with Z13Mad5+Anaxa (12%).

Next, cytokine secretion was assessed. To this end, C57BL/6 mice wereimplanted i.c. with 5×10⁵ Gl261-Quad tumor cells and vaccinated twice(d7 and 21) by s.c. injection of 2 nmol of Z13Mad5Anaxa or 2 nmol ofZ13Mad5 and 2 nmol of Anaxa. BILs were isolated and cultured during 6 hwith matured BMDCs loaded or not with SIINFEKL peptide in presence ofBrefeldinA before intracellular staining for cytokines. Results areshown in FIG. 35.

Despite heterogeneity, a high level of cytokine secretion was observedfor brain-infiltrating CD8 T cells from mice vaccinated withZ13Mad5Anaxa. These results demonstrate that Z13Mad5Anaxa vaccine wasable to elicit a stronger SIINFEKL specific CD8 T cell immune responsein the brain of tumor-bearing mice with potent effector function.

The results obtained are indicating that Z13Mad5Anaxa is efficacious foreliciting high brain infiltrating SIINFEKL-specific CD8 immune response.Z13Mad5Anaxa is able to promote the secretion of cytokine byantigen-specific CD8 T cells in the brain.

Example 21 Vaccine Efficacy on Survival in the Gl261-Quad GlioblastomaModel

In an independent experiment, the survival of control andZ13Mad5Anaxa-vaccinated mice was monitored. The therapeutic settingswere three consecutive vaccinations with 2 nmol of Z13Mad5Anaxa at day7, 21 and 35, post i.c. tumor implantation.

C57BL/6 mice were implanted i.c. with 5×10⁵ Gl261-Quad tumor cells andvaccinated three times (d7, d21 and d35) by s.c. injection of 2 nmol ofZ13Mad5Anaxa. Mice were weight daily and euthanized when weight lossreached more than 15%. Results are shown in FIG. 36.

The results show that Z13Mad5Anaxa therapeutic vaccination is moreefficacious than the control group with a median survival prolonged by10 days.

Example 22 Vaccine Efficacy in a Subcutaneous Tumor Model—ProphylacticSetting

This study is based on the results obtained in Example 10 as shown inFIGS. 17-19.

To evaluate the effect of the Anaxa-conjugated construct proteinsdesigned in Example 7 on tumor growth control, a benchmark tumor modelwas used, namely the s.c. implantation of EG.7-OVA thymoma cells. Incontrast to Example 10, wherein vaccination was performed on days 5 and13, in the present study a prophylactic setting was evaluated, whereinmice were vaccinated 21 and 7 days before tumor implantation.

C57BL/6 mice were vaccinated twice (d-21 and d-7) by s.c. injection of0.5 nmol of Z13Mad5Anaxa in the right flank and then implanted at day0s.c. with 3×10⁵ EG7-OVA tumor cells in the left flank and. Tumor sizewas measured with a caliper.

The results are shown in FIG. 37 with tumor volume (FIG. 37A) andsurvival rate (FIG. 37B). The data is showing that prophylacticvaccination with Z13Mad5Anaxa is highly efficacious for controllingtumor growth and survival rate. The volume of the tumor is highlysignificantly decreased in mice treated with Z13Mad5Anaxa as compared tocontrol mice. The survival rate is highly significantly increased inmice treated with Z13Mad5Anaxa as compared to control mice.

Example 23 Vaccine Efficacy in a Subcutaneous Tumor Model—TherapeuticSetting with Established Tumor

This study is based on the results obtained in Example 10 as shown inFIGS. 17-19 and on the results obtained in Example 22 shown in FIG. 37.It was the goal of this study to evaluate the effect of Z13Mad5Anaxa(cf. Example 7) on an established tumor.

For this purpose, the s.c. model of B16-OVA melanoma cells was used. Inthis model tumor cells are spreading slowly, therefore allowing a biggervaccination time window.

The first vaccination with the low dose of 0.5 nmol of Z13Mad5Anaxa wasperformed once the tumor was established and visible i.e. at day 14after tumor cell implantation. A second vaccination was done at day 21.

Thus, C57BL/6 mice were implanted s.c. with 1×10⁵ B16-OVA tumor cells inthe left flank and vaccinated twice (d14 and d21) by s.c. injection of0.5 nmol of Z13Mad5Anaxa in the right flank. Tumor growth and survivalcurves were monitored. Results are shown in FIG. 38.

The results indicate that Z13Mad5Anaxa efficaciously controls the growthof an established and visible tumor. Moreover, despite an establishedand visible tumor survival rates increased in mice treated withZ13Mad5Anaxa as compared to controls.

Example 24 Vaccine Efficacy in a Subcutaneous Tumor Model—TherapeuticSetting: Effect of the CPP

The protocol of this study corresponds to the study described in Example10, with the difference that an additional group “Mad5Anaxa” (cf.Example 16) was evaluated.

Briefly, a benchmark tumor model was used, namely the s.c. implantationof EG.7-OVA thymoma cells. C57BL/6 mice were implanted s.c. with 3×10⁵EG7-OVA tumor cells in the left flank. After tumor implantation, groupsof 7 mice each were vaccinated s.c. in the right flank at day 5 and 13by subcutaneous injection of 0.5 nmol of either Z13Mad5Anaxa (group 1)or Mad5Anaxa (group 2) and compared to a control group. Tumor size wasmeasured with a caliper. Results are shown in FIG. 39.

The results show that the mice treated with Z13Mad5Anaxa show asignificantly decreased tumor volume and a significantly increasedsurvival rate compared to both, control mice and mice treated withMad5Anaxa, i.e. a construct without CPP. These results indicate that thepresence of a CPP results in significantly decreased tumor volume and asignificantly increased survival rate, i.e. in increased efficiency ofvaccination. Therefore, the results indicate—together with the resultsobtained in Example 10—that the presence of a CPP and the TLR agonistexert a synergic effect on tumor growth and survival rate.

Example 25 Comparison of the Kinetic of Immune Responses with Complexeshaving Different Cell Penetrating Peptides

To investigate the effect of different CPPs in the complex according tothe present invention the fusion protein Z13Mad5Anaxa as described above(cf. Example 7) was used.

In addition, further fusion proteins were designed, which comprise CPPsother than Z13—namely Z14 (SEQ ID NO: 7) or Z18 (SEQ ID NO: 11). Thosefusion proteins also comprise the protein “MAD5”, which consists ofdifferent CD8⁺ and CD4⁺ epitopes from various antigens, and the TLR2peptide agonist “Anaxa”. Accordingly, the following constructs wereadditionally designed:

Z14Mad5Anaxa Sequence: (SEQ ID NO: 33) MHHHHHHKRY KNRVASRKSR AKFKQLLQHYREVAAAKESL KISQAVHAAH AEINEAGREV VGVGALKVPR NQDWLGVPRF AKFASFEAQGALANIAVDKA NLDVEQLESI INFEKLTEWT GSSTVHEILC KLSLEGDHST PPSAYGSVKPYTNFDAE Z18Mad5Anaxa Sequence: (SEQ ID NO: 34) MHHHHHHREV AAAKSSENDRLRLLLKESLK ISQAVHAAHA EINEAGREVV GVGALKVPRN QDWLGVPRFA KFASFEAQGALANIAVDKAN LDVEQLESII NFEKLTEWTG SSTVHEILCK LSLEGDHSTP PSAYGSVKPY TNFDAE

C57BL/6 mice were assigned to eight different groups (4 mice per group):three groups receiving 2 nmol of either Z13Mad5Anaxa, Z14Mad5Anaxa orZ18Mad5Anaxa and a respective control and three groups receiving 0.5nmol of Z13Mad5Anaxa, Z14Mad5Anaxa or Z18Mad5Anaxa and a respectivecontrol. The mice were vaccinated five times (Week0, Week2, Week4, Week6and Week8) s.c. Mice were bled 7 days after the 2^(nd), 3^(rd), 4^(th)and 5^(th) vaccination and multimer staining was performed (oneexperiment with 4 mice per group).

The results are shown in FIG. 40. All groups vaccinated withZ13Mad5Anaxa, Z14Mad5Anaxa or Z18Mad5Anaxa showed an increasedpercentage of multimer-positive cells compared to the control group(except for the second vaccination of Z18Mad5Anaxa). These resultsindicate that complexes according to the present invention havingdifferent cell penetrating peptides are able to elicit an immuneresponse at different doses.

Example 26 Comparison of T Cell Immune responses with Complexes havingDifferent Cell Penetrating Peptides

To investigate the CD8 T cell immune responses in more detail, C57BL/6mice were assigned to three different groups (3-4 mice per group):naïve, Z13Mad5Anaxa or Z14Mad5Anaxa.

C57BL/6 mice of the Z13Mad5Anaxa group and of the Z14Mad5Anaxa groupwere vaccinated five times (Week0, Week2, Week4, Week6 and Week8) s.c.with 2 nmol of either Z13Mad5Anaxa (cf. Example 7) or Z14Mad5Anaxa (cf.Example 25). Nine days after the 5^(th) vaccination, mice wereeuthanized, organs recovered and multimer staining was performed toidentify the percentage of SIINFEKL-specific CD8 T cells in the spleen,bone marrow and draining lymph nodes (inguinal and axillary).

The results are shown in FIG. 41. Mice vaccinated with Z13Mad5Anaxa orwith Z14Mad5Anaxa showed a similar increase in multimer-positive cells,in particular in the spleen and bone marrow as well as a slight increasein draining lymph nodes.

To further investigate the CD8 T cell effector function aftervaccination with complexes with different CPPs, in the same groups ofmice as described above Elispot assay was performed on spleen cellsstimulated with SIINFEKL OVACD8 peptide (SEQ ID NO: 35) nine days afterthe 5^(th) vaccination in order to quantify IFN-γ producing cells.

The results are shown in FIG. 42A. Mice vaccinated with Z13Mad5Anaxashowed a significant increase in IFN-γ producing cells compared to naïvemice. Mice vaccinated with Z14Mad5Anaxa showed also an increase in IFN-γproducing cells compared to naïve mice, however, the increase was notsignificant, which may be due to the low number of mice (3 mice inZ14Mad5Anaxa group).

To investigate the CD4 T cell responses after vaccination with complexeswith different CPPs, in the same groups of mice as described aboveElispot assay was performed on spleen cells stimulated with OVACD4peptide (SEQ ID NO: 36) nine days after the 5^(th) vaccination in orderto quantify IFN-γ producing cells.

The results are shown in FIG. 42B. Mice vaccinated with Z13Mad5Anaxashowed a highly significant increase in IFN-γ producing cells comparedto naïve mice. Mice vaccinated with Z14Mad5Anaxa showed also an increasein IFN-γ producing cells compared to naïve mice, however, the increasewas not significant, which may be due to the low number of mice (3 micein Z14Mad5Anaxa group).

In addition, in the above described groups of mice, intracellularstaining was performed on spleen cells stimulated with SIINFEKL OVACD8peptide (SEQ ID NO: 35) to identify CD107a⁺IFN-γ⁺TNF-α⁺ cells. Resultsare shown in FIG. 43. Mice vaccinated with Z13Mad5Anaxa or withZ14Mad5Anaxa showed a similar increase in CD107a⁺IFN-γ⁺TNF-α⁺ cells.

Example 27 Comparison of the Effect of Complexes having Different CellPenetrating Peptides on Tumor Growth and Survival in the EG.7-OVA s.c.Model

To investigate the effects of complexes having different cellpenetrating peptides on tumor growth and survival the EG.7-OVA s.c.model was used. On d0 C57BL/6 mice were implanted s.c. with 3×10⁵EG7-OVA tumor cells in the left flank and assigned to three differentgroups (naïve, Z13Mad5Anaxa and Z14Mad5Anaxa). Mice were vaccinatedtwice at d5 and d13 after tumor implantation by s.c. injection of either0.5 nmol of Z13Mad5Anaxa or Z14Mad5Anaxa in the right flank.

Results are shown in FIG. 44. Vaccination with Z13Mad5Anaxa or withZ14Mad5Anaxa resulted in significantly decreased tumor volumes comparedto control mice (FIG. 44A) as well as to significantly increasedsurvival rates compared to control mice (FIG. 44B). Those resultsindicate that both complexes, Z13Mad5Anaxa and Z14Mad5Anaxa, are able tosignificantly decrease tumor growth and to significantly prolongsurvival.

Example 28 Comparison of the Immune responses after Vaccination withComplexes having Different cell Penetrating Peptides

In this experiment the effect of different CPPs in the complex accordingto the present invention was investigated by using a complex with theTLR agonist “EDA”. Therefore, the fusion protein EDAZ13Mad5 as describedabove (cf. Example 1) was used.

In addition, further fusion proteins were designed, which comprise CPPsother than Z13—namely Z14 (SEQ ID NO: 7) or Z18 (SEQ ID NO: 11). Thosefusion proteins also comprise the protein “MAD5”, which consists ofdifferent CD8⁺ and CD4⁺ epitopes from various antigens, and the TLR4peptide agonist “EDA”. Accordingly, the following constructs wereadditionally designed:

EDAZ14Mad5 Sequence: (SEQ ID NO: 37) MHHHHHHNID RPKGLAFTDV DVDSIKIAWESPQGQVSRYR VTYSSPEDGI RELFPAPDGE DDTAELQGLR PGSEYTVSVV ALHDDMESQPLIGIQSTKRY KNRVASRKSR AKFKQLLQHY REVAAAKESL KISQAVHAAH AEINEAGREVVGVGALKVPR NQDWLGVPRF AKFASFEAQG ALANIAVDKA NLDVEQLESI INFEKLTEWT GSEDAZ18Mad5 Sequence: (SEQ ID NO: 38) MHHHHHHNID RPKGLAFTDV DVDSIKIAWESPQGQVSRYR VTYSSPEDGI RELFPAPDGE DDTAELQGLR PGSEYTVSVV ALHDDMESQPLIGIQSTREV AAAKSSENDR LRLLLKESLK ISQAVHAAHA EINEAGREVV GVGALKVPRNQDWLGVPRFA KFASFEAQGA LANIAVDKAN LDVEQLESII NFEKLTEWTG S

C57BL/6 mice were assigned to eight different groups (4 mice per group):three groups receiving 2 nmol of either EDAZ13Mad5, EDAZ14Mad5 orEDAZ18Mad5 and a respective control and three groups receiving 0.5 nmolof either EDAZ13Mad5, EDAZ14Mad5 or EDAZ18Mad5 and a respective controlgroup. The mice were vaccinated three times (WeekO, Week2 and Week4)s.c. Mice were bled 7 days after the 2^(nd) and 3^(rd) vaccination andmultimer staining was performed (one experiment with 4 mice per group).

The results are shown in FIG. 45. All groups vaccinated with EDAZ13Mad5,EDAZ14Mad5 or EDAZ18Mad5 showed an increased percentage ofmultimer-positive cells compared to the control group. These resultsindicate that complexes according to the present invention havingdifferent cell penetrating peptides are able to elicit an immuneresponse at different doses.

Example 29 Effect of EDAZ14Mad5 on Tumor Growth and Survival in theEG.7-OVA s.c. Model

To investigate the effect of EDAZ14Mad5 on tumor growth and survival theEG.7-OVA s.c. model was used (cf. Example 4 and FIGS. 9-11 for theeffect of EDAZ13Mad5 in the same model).

On d0 C57BL/6 mice were implanted s.c. with 3×10⁵ EG7-OVA tumor cells inthe left flank and assigned to two different groups (naïve andEDAZ14Mad5). Mice were vaccinated twice at d5 and d13 after tumorimplantation by s.c. injection of 0.5 nmol of EDAZ14Mad5 in the rightflank.

Results are shown in FIG. 46. Similarly to EDAZ13Mad5 (cf. Example 4,FIGS. 9-11) vaccination with EDAZ14Mad5 resulted in significantlydecreased tumor volumes compared to control mice (FIG. 46A) as well asto significantly increased survival rates compared to control mice (FIG.46B). Those results indicate that EDAZ14Mad5 is able to significantlydecrease tumor growth and to significantly prolong survival—similarly toEDAZ13Mad5 (cf. Example 4, FIGS. 9-11).

Example 30 Superior Efficacy of Z13Mad5Anaxa Fusion Construct comparedto Z13Mad5 and Anaxa in a Glioblastoma Model

To investigate the efficacy of a complex according to the presentinvention the glioblastoma model was chosen (cf. Example 20). Namely,Z13Mad5Anaxa (cf. Example 7; SEQ ID NO: 28) was administered to onegroup of mice, whereas Z13Mad5 (SEQ ID NO: 29) and Anaxa (SEQ ID NO: 15)were administered (both together) to another group of mice.

T cell homing at the tumor site was analyzed in Gl261-Quad tumor-bearingmice (7-16 mice per group) vaccinated twice, namely at day 7 and at day21 after tumor implantation (day 0), with 2 nmol Z13Mad5Anaxa vaccine. Agroup vaccinated with both, Z13Mad5 and Anaxa (equimolar toZ13Mad5Anaxa), was used as control. Briefly, C57BL/6 mice were implantedi.c. (intracranially) with 5×10⁵ Gl261-Quad tumor cells and vaccinatedtwice (at d7 and d21 following implantation) by s.c. injection of 2 nmolof Z13Mad5Anaxa (group 1) or 2 nmol of Z13Mad5 and 2 nmol of Anaxa(group 2). At day 28, the blood and the brain infiltrating leukocytes(BILs) were analyzed, whereby SIINFEKL-specific CD8 T cells werequantified in blood and in BILs at d28 by multimer staining (7-16 miceper group).

Results are shown in FIG. 47. A significantly higher percentage ofSIINFEKL-specific CD8 T cells was observed in the blood ofZ13Mad5Anaxa-vaccinated mice as compared to mice vaccinated with both,Z13Mad5 and Anaxa (FIG. 47A). Similarly, a stronger accumulation ofSIINFEKL-specific CD8 T cells was observed in the BILs ofZ13Mad5Anaxa-vaccinated mice as compared to mice vaccinated with Z13Mad5and Anaxa separately (FIG. 47B, p=0.0539).

Next, cytokine secretion was assessed. To this end, C57BL/6 mice wereimplanted i.c. with 5×10⁵ Gl261-Quad tumor cells and vaccinated twice(d7 and 21) by s.c. injection of 2 nmol of Z13Mad5Anaxa or 2 nmol ofZ13Mad5 and 2 nmol of Anaxa. BILs were isolated and cultured during 6 hwith matured BMDCs loaded or not with SIINFEKL peptide (SEQ ID NO: 35)in presence of BrefeldinA before intracellular staining for cytokines.

Results are shown in FIG. 48. In general, a high level of cytokinesecretion was observed for brain-infiltrating CD8 T cells from micevaccinated with Z13Mad5Anaxa. In particular, a significantly highersecretion of total IFN-γ and of IFN-γ and TNF-α together was observedfor brain-infiltrating CD8 T cells from mice vaccinated withZ13Mad5Anaxa as compared to mice vaccinated with Z13Mad5 and Anaxaseparately.

Taken together, these results demonstrate that Z13Mad5Anaxa vaccine (ascompared to Z13Mad5 and Anaxa administered separately) was able toelicit a stronger SIINFEKL specific CD8 T cell immune response in thebrain of tumor-bearing mice with potent effector function.

The results obtained are indicating that Z13Mad5Anaxa is efficacious foreliciting high brain infiltrating SIINFEKL-specific CD8 immune response.Z13Mad5Anaxa is able to promote the secretion of cytokine byantigen-specific CD8 T cells in the brain.

Example 31 Effect of another Antigenic Cargo in the Complex according tothe Present Invention

To investigate the effect of a different antigenic cargo (“Mad8”),another complex comprising a cell penetrating peptide, differentantigens and a TLR peptide agonist was designed (“Z13Mad8Anaxa”).Z13Mad8Anaxa differs from Z13Mad5Anaxa (described in Example 7) in theantigenic cargoes. In particular, “Z13Mad8Anaxa” is a fusion proteincomprising the cell-penetrating peptide “Z13”, the antigenic cargo“MAD8” comprising CD8 and CD4 epitopes of glycoprotein 70, and the TLRpeptide agonist “Anaxa”. In the following, the amino acid sequence ofZ13Mad8Anaxa is shown with the cell-penetrating peptide “Z13” shownunderlined and the TLR peptide agonist “Anaxa” shown in italics:

(SEQ ID NO: 39) KRYKNRVASR KSRAKFKQLL QHYREVAAAK SSENDRLRLLLK VTYHSPSYAYHQFERRAILN RLVQFIKDRI SVVQALVLTS TVHEILCKLS LEGDHSTPPS AYGSVKPYTN FDAE

Naïve Balb/c mice (4 mice per group) were vaccinated four times s.c.(week0, week2, week4 and week6 with 2 nmol of Z13Mad8Anaxa.

To investigate the CD4 T cell responses after vaccination, one weekafter the 4^(th) vaccination, mice were euthanized; organs recovered andex vivo Elispot assay was performed on spleen cells stimulated withgp70CD4 peptide (SEQ ID NO: 64) or gp70CD8 peptide (SEQ ID NO: 65) inorder to quantify IFN-γ-producing epitope-specific CD4 and CD8 T cells.

The results are shown in FIG. 49. Mice vaccinated with Z13Mad8Anaxashowed a significant increase in IFN-γ-producing cells compared to naïvemice. These data show that Z13Mad8Anaxa vaccine was able to elicitpotent epitope-specific CD8 and CD4 T cell immune response and thus thatthe complex according to the present invention is able to elicitself-antigen immune response.

Example 32 Effect of another Antigenic Cargo in the Complex according tothe Present Invention

To investigate the effect of a further different antigenic cargo(“Mad11”), another complex comprising a cell penetrating peptide,different antigens and a TLR peptide agonist was designed(“Z13Mad11Anaxa”). Z13Mad11Anaxa differs from Z13Mad5Anaxa (described inExample 7) in the antigenic cargoes. In particular, “Z13Mad11Anaxa” is afusion protein comprising the cell-penetrating peptide “Z13”, theantigenic cargo “MAD11” comprising two CD8 epitopes of surviving asdescribed in Derouazi M, Wang Y, Marlu R, et al. Optimal epitopecomposition after antigen screening using a live bacterial deliveryvector: Application to TRP-2. Bioengineered Bugs. 2010; 1(1):51-60.doi:10.4161/bbug.1.1.9482, and the TLR peptide agonist “Anaxa”. In thefollowing, the amino acid sequence of Z13Mad11Anaxa is shown with thecell-penetrating peptide “Z13” shown underlined and the TLR peptideagonist “Anaxa” shown in italics:

(SEQ ID NO: 40) KRYKNRVASRKSRAKFKQLLQHYREVAAAKSSENDRLRLLLKNYRIATFKNWPFLEDCAMEELTVSEFLKLDRQRSTVHEILCKLSLEGDHSTP PSAYGSVKPYTNFDAE

Naïve C57BL/6 mice (5 mice per group) were implanted i.v. with 1×10⁵ B16melanoma tumor cells and vaccinated twice (d0 and d10) by subcutaneousinjection of 1 nmol of Z13Mad11Anaxa. On day18 mice were euthanized,organs recovered and ex vivo Elispot assay was performed on spleen cellsstimulated with survivin peptides survivin20-28 (SEQ ID NO: 67) andsurvivin97-104: (SEQ ID NO: 68) in order to quantify IFN-γ producingsurvivin-specific T cells.

The results are shown in FIG. 50. Mice vaccinated with Z13Mad11Anaxashowed less metastasis compared to naïve mice (FIG. 50A). Moreover, inthe spleen of mice vaccinated with Z13Mad11Anaxa significantly highernumbers of IFN-γ producing survivin-specific T cells were observed (FIG.49B).

The results obtained show that Z13Mad11Anaxa is efficacious for reducingthe number of metastasis and Z13Mad11Anaxa is able to promote thesecretion of cytokines by antigen-specific CD8 T cells in the spleen.

Example 33 Effect of another Antigenic Cargo in the Complex according tothe Present Invention

To investigate the effect of a further different antigenic cargo(“Mad9”), another complex comprising a cell penetrating peptide, adifferent antigen and a TLR peptide agonist was designed(“Z13Mad9Anaxa”). Z13Mad9Anaxa differs from Z13Mad5Anaxa (described inExample 7) in the antigenic cargo. In particular, “Z13Mad9Anaxa” is afusion protein comprising the cell-penetrating peptide “Z13”, theantigenic cargo “Mad9” comprising the neoantigen as identified by Yadavet al. Nature. 2014 Nov. 27; 515(7528):572-6 from MC-38 tumor cell line,and the TLR peptide agonist “Anaxa”. In the following, the amino acidsequence of Z13Mad9Anaxa is shown with the cell-penetrating peptide“Z13” shown underlined and the TLR peptide agonist “Anaxa” shown initalics:

(SEQ ID NO: 41) KRYKNRVASRKSRAKFKQLLQHYREVAAAKSSENDRLRLLLKHLELASMTNMELMSSIVSTVHEILCKLSLEGDHSTPPSAYGSVKPYTNFDAE

Naïve C57BL/6 mice (4 mice per group) were vaccinated four times s.c.(week0, week2, week4 and week6 with 2 nmol of Z13Mad9Anaxa. Toinvestigate the CD8 T cell responses after vaccination, one week afterthe 4^(th) vaccination, mice were euthanized, organs recovered andElispot assay was performed on spleen cells after a 7-day in vitrorestimulation with stimulated with adpgk peptide (SEQ ID NO: 66) inorder to quantify to quantify IFN-γ-producing epitope-specific CD8 Tcells.

The results are shown in FIG. 51. Mice vaccinated with Z13Mad9Anaxashowed a significant increase in effector neoantigen-specific CD8 Tcells compared to naïve mice.

Example 34 Comparison of the Immune responses after Vaccination withComplexes having Different Cell Penetrating Peptides

In this experiment the effect of a further different CPP in the complexaccording to the present invention was investigated by using a complexwith the TLR agonist “Anaxa”. Therefore, the fusion protein Z13Mad5Anaxaas described above (cf. Example 7, SEQ ID NO: 28) was used.

In addition, a further fusion protein was designed, which comprise theTAT CPP combined to furin linkers as described in Lu et al.,Multiepitope trojan antigen peptide vaccines for the induction ofantitumor CTL and Th immune responses J. Immunol., 172 (2004), pp.4575-4582. That fusion protein also comprises the protein “MAD5”, whichconsists of different CD8⁺ and CD4⁺ epitopes from various antigens, andthe TLR4 peptide agonist “Anaxa”. Accordingly, the following constructwas additionally designed:

TatFMad5Anaxa Sequence: (SEQ ID NO: 46)RKKRRQRRRRVKRISQAVHAAHAEINEAGRRVKRKVPRNQDWLRVKRASFEAQGALANIAVDKARVKRSIINFEKLRVKRSTVHEILCKLSLEGDHS TPPSAYGSVKPYTNFDAE

C57BL/6 mice were assigned to three different groups (8 mice per group):one group receiving 2 nmol of Z13Mad5Anaxa, one group receiving 2 nmolof TatFMad5Anaxa and a respective control. The mice were vaccinated twotimes (Week0 and Week2) s.c. with either 2 nmol of Z13Mad5Anaxa or 2nmol of TatFMad5Anaxa. Mice were bled 7 days after the 2^(nd)vaccination and multimer staining was performed (8 mice per group).

The results are shown in FIG. 52. Mice vaccinated with Z13Mad5Anaxa orTatFMad5Anaxa showed an increased percentage of multimer-positive cellscompared to the control group. These results indicate that complexesaccording to the present invention having different cell penetratingpeptides are able to elicit an immune response at different doses.However, the CPP derived from ZEBRA (Z13) was better than the TAT CPP.

Example 35 Superior Efficacy of Z13Mad5Anaxa Fusion Construct comparedto Z13Mad5 and Anaxa in Naïve Mice

Next, the efficacy of a complex according to the present invention wasinvestigated in naïve mice. Namely, Z13Mad5Anaxa (cf. Example 7; SEQ IDNO: 28) was administered to one group of mice, whereas Z13Mad5 (SEQ IDNO: 29) and Anaxa (SEQ ID NO: 15) were administered (both together) toanother group of mice.

C57BL/6 mice of the Z13Mad5Anaxa group and of the Z13Mad5+Anaxa groupwere vaccinated once (Week0) by s.c. injection of 2 nmol of Z13Mad5Anaxa(group 1) or 2 nmol of Z13Mad5 and 2 nmol of Anaxa (group 2). At day 14,the blood was analyzed, whereby SIINFEKL-specific CD8 T cells werequantified in blood by multimer staining (4-8 mice per group).

Results are shown in FIG. 53. A significantly higher percentage ofSIINFEKL-specific CD8 T cells was observed in the blood ofZ13Mad5Anaxa-vaccinated mice as compared to mice vaccinated with Z13Mad5and Anaxa separately (FIG. 53).

Taken together, these results demonstrate that Z13Mad5Anaxa vaccine (ascompared to Z13Mad5 and Anaxa administered separately) was able toelicit a stronger SIINFEKL specific CD8 T cell immune response in theperiphery.

Example 36 Effect of another Antigenic Cargo in the Complex according tothe Present Invention

To investigate the effect of a further different antigenic cargo(“Mad12”), another complex comprising a cell penetrating peptide, adifferent antigen and a TLR peptide agonist was designed(“Z13Mad12Anaxa”). Z13Mad12Anaxa differs from Z13Mad5Anaxa (described inExample 7) in the antigenic cargo. In particular, “Z13Mad12Anaxa” is afusion protein comprising the cell-penetrating peptide “Z13”, theantigenic cargo “MAD12” comprising three neoantigens as identified byYadav et al. Nature. 2014 Nov. 27; 515(7528):572-6 from MC-38 tumor cellline, and the TLR peptide agonist “Anaxa”. In the following, the aminoacid sequence of Z13Mad12Anaxa is shown:

(SEQ ID NO: 69) KRYKNRVASRKSRAKFKQLLQHYREVAAAKSSENDRLRLLLKLFRAAQLANDVVLQIMEHLELASMTNMELMSSIVVISASIIVFNLLELEGSTVHEILCKLSLEGDHSTPPSAYGSVKPYTNFDAENaïve C57BL/6 mice (4 mice per group) were vaccinated twice s.c. (week0,week2) with 2 nmol of Z13Mad12Anaxa. To investigate the CD8 T cellresponses after vaccination, one week after the 2^(nd) vaccination, theblood was analyzed, whereby neoantigen reps1-specific CD8 T cells werequantified in blood by multimer staining (4 mice per group).

The results are shown in FIG. 54. Mice vaccinated with Z13Mad12Anaxashowed a significant increase in effector neoantigen-specific CD8 Tcells compared to naïve mice.

Example 37 In Vitro Human Dendritic Cell Maturation

The goal of this study was to investigate the capacity of a complex foruse according to the present invention (“Z13Mad5Anaxa”, SEQ ID NO: 28,cf. Example 7) to induce maturation of dendritic cells in comparison toa complex lacking a TLR peptide agonist (“Z13Mad5”, SEQ ID NO: 29, cf.Example 1).

The Z13Mad5Anaxa polypeptide and the Z13Mad5 polypeptide wereinvestigated for their capacity to induce human dendritic cell (DC)maturation. After incubation over night with 300 nM of protein,activation markers expression (CD86, CD80, CD83 and HLA-DR) was assessedon the human DCs by FACS (FIG. 55). Same buffer volumes of each proteinwere used as negative controls.

Results are shown in FIG. 55. Whereas Z13Mad5Anaxa induced maturation ofhuman DCs, shown by the up-regulation of CD86, HLADR and CD83, Z13Mad5was not able to activate human DCs. These results indicate that theAnaxa portion of the protein is responsible for the up-regulation of theactivation markers on the human DCs.

TABLE OF SEQUENCES AND SEQ ID NUMBERS (SEQUENCE LISTING): SEQ ID NOSequence Remarks SEQ ID NO: 1 RQIKIYFQNRRMKWKK CPP: Penetratin SEQ IDNO: 2 YGRKKRRQRRR CPP: TAT minimal SEQ ID NO: 3MMDPNSTSEDVKFTPDPYQVPFVQAFDQATRV ZEBRA amino acidYQDLGGPSQAPLPCVLWPVLPEPLPQGQLTAY sequence (naturalHVSTAPTGSWFSAPQPAPENAYQAYAAPQLFP sequence fromVSDITQNQQTNQAGGEAPQPGDNSTVQTAA Epstein - Barr virusAVVFACPGANQGQQLADIGVPQPAPVAAPAR (EBV)) (YP_401673)RTRKPQQPESLEECDSELEIKRYKNRVASRKCRAK FKQLLQHYREVAAAKSSENDRLRLLLKQMCPSLDVDSIIPRTPDVLHEDLLNF SEQ ID NO: 4 KRYKNRVASRKCRAKFKQLLQHYREVAAAKSSE CPP1(Z11) NDRLRLLLKQMC SEQ ID NO: 5 KRYKNRVASRKCRAKFKQLLQHYREVAAAKSSE CPP2(Z12) NDRLRLLLK SEQ ID NO: 6 KRYKNRVASRKSRAKFKQLLQHYREVAAAKSSE CPP3(Z13) NDRLRLLLK SEQ ID NO: 7 KRYKNRVASRKSRAKFKQLLQHYREVAAAK CPP4 (Z14)SEQ ID NO: 8 KRYKNRVASRKSRAKFK CPP5 (Z15) SEQ ID NO: 9 QHYREVAAAKSSENDCPP6 (Z16) SEQ ID NO: 10 QLLQHYREVAAAK CPP7 (Z17) SEQ ID NO: 11REVAAAKSSENDRLRLLLK CPP8 (Z18) SEQ ID NO: 12 KRYKNRVA CPP9 (Z19) SEQ IDNO: 13 VASRKSRAKFK CPP10 (Z20) SEQ ID NO: 14ESLKISQAVHAAHAEINEAGREVVGVGAL MAD5 cargo KVPRNQDWLGVPRFAKFASFEAQGALANIAVDKANLDVEQLESIINFEKLTEWTGS SEQ ID NO: 15STVHEILCKLSLEGDHSTPPSAYGSVKPYTNFDAE TLR2 peptide agonist Anaxa SEQ IDNO: 16 DDDK enterokinase target site SEQ ID NO: 17 IEDGR factor Xatarget site SEQ ID NO: 18 LVPRGS thrombin target site SEQ ID NO: 19ENLYFQG protease TEV target site SEQ ID NO: 20 LEVLFQGP PreScissionprotease target SEQ ID NO: 21 RX(R/K)R furin target site SEQ ID NO: 22GGGGG peptidic linker SEQ ID NO: 23 GGGG peptidic linker SEQ ID NO: 24EQLE peptidic linker SEQ ID NO: 25 TEWT peptidic linker SEQ ID NO: 26MHHHHHHNIDRPKGLAFTDVDVDSIKIA EDAZ13Mad5 WESPQGQVSRYRVTYSSPEDGIRELFPAPDGEDDTAELQGLRPGSEYTVSVVALHDD MESQPLIGIQSTKRYKNRVASRKSRAKFKQLLQHYREVAAAKSSENDRLRLLLKESLKISQ AVHAAHAEINEAGREVVGVGALKVPRNQDWLGVPRFAKFASFEAQGALANIAVDK ANLDVEQLESIINFEKLTEWTGS SEQ ID NO: 27MHHHHHHSTVHEILCKLSLEGDHSTPPSA AnaxaZ13Mad5 YGSVKPYTNFDAEKRYKNRVASRKSRAKFKQLLQHYREVAAAKSSENDRLRLLLKESLKI SQAVHAAHAEINEAGREVVGVGALKVPRNQDWLGVPRFAKFASFEAQGALANIAVD KANLDVEQLESIINFEKLTEWTGS SEQ ID NO: 28MHHHHHHKRYKNRVASRKSRAKFKQLL Z13Mad5Anaxa QHYREVAAAKSSENDRLRLLLKESLKISQAVHAAHAEINEAGREVVGVGALKVPRNQD WLGVPRFAKFASFEAQGALANIAVDKANLDVEQLESIINFEKLTEWTGSSTVHEILCKLSL EGDHSTPPSAYGSVKPYTNFDAE SEQ ID NO: 29MHHHHHHKRYKNRVASRKSRAKFKQLL Z13Mad5 QHYREVAAAKSSENDRLRLLLKESLKISQAVHAAHAEINEAGREVVGVGALKVPRNQD WLGVPRFAKFASFEAQGALANIAVDKANLDVEQLESIINFEKLTEWTGS SEQ ID NO: 30 MHHHHHHESLKISQAVHAAHAEINEAGREV Mad5VGVGALKVPRNQDWLGVPRFAKFASFEAQ GALANIAVDKANLDVEQLESIINFEKLTEWTGS SEQ IDNO: 31 MHHHHHHNIDRPKGLAFTDVDVDSIKIA EdaMad5WESPQGQVSRYRVTYSSPEDGIRELFPAP DGEDDTAELQGLRPGSEYTVSVVALHDDMESQPLIGIQSTESLKISQAVHAAHAEINE AGREVVGVGALKVPRNQDWLGVPRFAKFASFEAQGALANIAVDKANLDVEQLESIIN FEKLTEWTGS SEQ ID NO: 32MHHHHHHESLKISQAVHAAHAEINEAG Mad5Anaxa REVVGVGALKVPRNQDWLGVPRFAKFASFEAQGALANIAVDKANLDVEQLESIINFEK LTEWTGSSTVHEILCKLSLEGDHSTPPSAYGSVKPYTNFDAE SEQ ID NO: 33 MHHHHHHKRYKNRVASRKSRAKFKQLL Z14 Mad5AnaxaQHYREVAAAKESLKISQAVHAAHAEINE AGREVVGVGALKVPRNQDWLGVPRFAKFASFEAQGALANIAVDKANLDVEQLESI INFEKLTEWTGSSTVHEILCKLSLEGDHSTPPSAYGSVKPYTNFDAE SEQ ID NO: 34 MHHHHHHREVAAAKSSENDRLRLLLKES Z18Mad5Anaxa LKISQAVHAAHAEINEAGREVVGVGALKV PRNQDWLGVPRFAKFASFEAQGALANIAVDKANLDVEQLESIINFEKLTEWTGSSTVH EILCKLSLEGDHSTPPSAYGSVKPYTNFDAE SEQ IDNO: 35 SIINFEKL SIINFEKL OVACD8 SEQ ID NO: 36 ISQAVHAAHAEINEAGR OVACD4peptide SEQ ID NO: 37 MHHHHHHNIDRPKGLAFTDVDVDSIKIA EDAZ14Mad5WESPQGQVSRYRVTYSSPEDGIRELFPAP DGEDDTAELQGLRPGSEYTVSVVALHDDMESQPLIGIQSTKRYKNRVASRKSRAKFKQ LLQHYREVAAAKESLKISQAVHAAHAEINEAGREVVGVGALKVPRNQDWLGVPRFA KFASFEAQGALANIAVDKANLDVEQLESII NFEKLTEWTGSSEQ ID NO: 38 MHHHHHHNIDRPKGLAFTDVDVDSIKIA EDAZ18Mad5WESPQGQVSRYRVTYSSPEDGIRELFPAP DGEDDTAELQGLRPGSEYTVSVVALHDDMESQPLIGIQSTREVAAAKSSENDRLRLLL KESLKISQAVHAAHAEINEAGREVVGVGALKVPRNQDWLGVPRFAKFASFEAQGALA NIAVDKANLDVEQLESIINFEKLTEWTGS SEQ ID NO: 39KRYKNRVASRKSRAKFKQLLQHYREVAAA Z13Mad8AnaxaKSSENDRLRLLLKVTYHSPSYAYHQFERRA ILNRLVQFIKDRISVVQALVLTSTVHEILCKLSLEGDHSTPPSAYGSVKPYTN FDAE SEQ ID NO: 40KRYKNRVASRKSRAKFKQLLQHYREVAAAKSSEN Z13Mad11AnaxaDRLRLLLKNYRIATFKNWPFLEDCAMEELTVSEFL KLDRQRSTVHEILCKLSLEGDHSTPPSAYGSVKPYTNFDAE SEQ ID NO: 41 KRYKNRVASRKSRAKFKQLLQHYREVAAAKSSEN Z13Mad9AnaxaDRLRLLLKHLELASMTNMELMSSIVSTVHEILCKLS LEGDHSTPPSAYGSVKPYTNFDAE SEQ ID NO:42 HLELASMTNMELMSSIV Mad9 SEQ ID NO: 43 VTYHSPSYAYHQFERRAILN Mad8 SEQ IDNO: 44 NYRIATFKNWPFLEDCAMEELTVSEFLKLDRQR Mad11 SEQ ID NO: 45NIDRPKGLAFTDVDVDSIKIAWESPQGQVSRYR EDAVTYSSPEDGIRELFPAPDGEDDTAELQGLRPGSEY TVSVVALHDDMESQPLIGIQST SEQ ID NO: 46RKKRRQRRRRVKRISQAVHAAHAEINEAGRRVK TatFMad5AnaxaRKVPRNQDWLRVKRASFEAQGALANIAVDKAR VKRSIINFEKLRVKRSTVHEILCKLSLEGDHSTPPSAYGSVKPYTNFDAE SEQ ID NO: 47 MAPPQVLAFGLLLAAATATFAAAQEECVCENYK EpCAMLAVNCFVNNNRQCQCTSVGAQNTVICSKLAAK CLVMKAEMNGSKLGRRAKPEGALQNNDGLYDPDCDESGLFKAKQCNGTSMCWCVNTAGVRRT DKDTEITCSERVRTYWIIIELKHKAREKPYDSKSLRTALQKEITTRYQLDPKFITSILYENNVITIDLVQNS SQKTQNDVDIADVAYYFEKDVKGESLFHSKKMDLTVNGEQLDLDPGQTLIYYVDEKAPEFSMQGL KAGVIAVIVVVVIAVVAGIVVLVISRKKRMAKYEKAEIKEMGEMHRELNA SEQ ID NO: 48 GLKAGVIAV EpCAM epitope SEQ ID NO: 49MTPGTQSPFFLLLLLTVLTVVTGSGHASSTPGGE MUC-1KETSATQRSSVPSSTEKNAVSMTSSVLSSHSPGSG SSTTQGQDVTLAPATEPASGSAATWGQDVTSVPVTRPALGSTTPPAHDVTSAPDNKPAPGSTAPPA HGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSA PDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAP GSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPA HGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSA PDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAP GSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPA HGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSA PDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAP GSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPA HGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSA PDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAP GSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPAHGVTSAPDTRPAPGSTAPPA HGVTSAPDNRPALGSTAPPVHNVTSASGSASGSASTLVHNGTSARATTTPASKSTPFSIPSHHSDTPT TLASHSTKTDASSTHHSSVPPLTSSNHSTSPQLSTGVSFFFLSFHISNLQFNSSLEDPSTDYYQELQRDIS EMFLQIYKQGGFLGLSNIKFRPGSVVVQLTLAFREGTINVHDVETQFNQYKTEAASRYNLTISDVSVS DVPFPFSAQSGAGVPGWGIALLVLVCVLVALAIVYLIALAVCQCRRKNYGQLDIFPARDTYHPMSEYP TYHTHGRYVPPSSTDRSPYEKVSAGNGGSSLSYTNPAVAATSANL SEQ ID NO: 50 GSTAPPVHN MUC-1 epitope SEQ ID NO: 51TAPPAHGVTS MUC-1 epitope SEQ ID NO: 52 MGAPTLPPAWQPFLKDHRISTFKNWPFLEGCACsurvivin TPERMAEAGFIHCPTENEPDLAQCFFCFKELEGWEPDDDPIEEHKKHSSGCAFLSVKKQFEELTLGEFL KLDRERAKNKIAKETNNKKKEFEETAKKVRRAIEQLAAMD SEQ ID NO: 53 RISTFKNWPF survivin epitope SEQ ID NO: 54MESPSAPPHRWCIPWQRLLLTASLLTFWNPPTTA CEAKLTIESTPFNVAEGKEVLLLVHNLPQHLFGYSWY KGERVDGNRQIIGYVIGTQQATPGPAYSGREIIYPNASLLIQNIIQNDTGFYTLHVIKSDLVNEEATG QFRVYPELPKPSISSNNSKPVEDKDAVAFTCEPETQDATYLWWVNNQSLPVSPRLQLSNGNRTLTLF NVTRNDTASYKCETQNPVSARRSDSVILNVLYGPDAPTISPLNTSYRSGENLNLSCHAASNPPAQYS WFVNGTFQQSTQELFIPNITVNNSGSYTCQAHNSDTGLNRTTVTTITVYAEPPKPFITSNNSNPVED EDAVALTCEPEIQNTTYLWWVNNQSLPVSPRLQLSNDNRTLTLLSVTRNDVGPYECGIQNKLSVDH SDPVILNVLYGPDDPTISPSYTYYRPGVNLSLSCHAASNPPAQYSWLIDGNIQQHTQELFISNITEKNS GLYTCQANNSASGHSRTTVKTITVSAELPKPSISSNNSKPVEDKDAVAFTCEPEAQNTTYLWWVNG QSLPVSPRLQLSNGNRTLTLFNVTRNDARAYVCGIQNSVSANRSDPVTLDVLYGPDTPIISPPDSSYL SGANLNLSCHSASNPSPQYSWRINGIPQQHTQVLFIAKITPNNNGTYACFVSNLATGRNNSIVKSIT VSASGTSPGLSAGATVGIMIGVLVGVAL SEQ IDNO: 55 YLSGANLNLS CEA epitope SEQ ID NO: 56 SWRINGIPQQ CEA epitope SEQID NO: 57 MTEYKLVVVGAGGVGKSALTIQLIQNHFVDEYD Kirsten RasPTIEDSYRKQVVIDGETCLLDILDTAGQEEYSAM RDQYMRTGEGFLCVFAINNTKSFEDIHHYREQIKRVKDSEDVPMVLVGNKCDLPSRTVDTKQAQDL ARSYGIPFIETSAKTRQRVEDAFYTLVREIRQYRLKKISKEEKTPGCVKIKKCIIM SEQ ID NO: 58 VVVGAGGVG Kirsten Ras epitope SEQ IDNO: 59 MPLEQRSQHCKPEEGLEARGEALGLVGAQAPAT MAGE-A3EEQEAASSSSTLVEVTLGEVPAAESPDPPQSPQGA SSLPTTMNYPLWSQSYEDSSNQEEEGPSTFPDLESEFQAALSRKVAELVHFLLLKYRAREPVTKAEMLGS VVGNWQYFFPVIFSKAFSSLQLVFGIELMEVDPIGHLYIFATCLGLSYDGLLGDNQIMPKAGLLIIVLAII AREGDCAPEEKIWEELSVLEVFEGREDSILGDPKKLLTQHFVQENYLEYRQVPGSDPACYEFLWGPRA LVETSYVKVLHHMVKISGGPHISYPPLHEWVLRE GEESEQ ID NO: 60 KVAELVHFL MAGE-A3 epitope SEQ ID NO: 61MAFVCLAIGCLYTFLISTTFGCTSSSDTEIKVNPPQ IL13Ralpha2DFEIVDPGYLGYLYLQWQPPLSLDHFKECTVEYE LKYRNIGSETWKTIITKNLHYKDGFDLNKGIEAKIHTLLPWQCTNGSEVQSSWAETTYWISPQGIPET KVQDMDCVYYNWQYLLCSWKPGIGVLLDTNYNLFYWYEGLDHALQCVDYIKADGQNIGCRFPY LEASDYKDFYICVNGSSENKPIRSSYFTFQLQNIVKPLPPVYLTFTRESSCEIKLKWSIPLGPIPARCFDYEIEIREDDTTLVTATVENETYTLKTTNETRQLCFVVR SKVNIYCSDDGIWSEWSDKQCWEGEDLSKKTLLRFWLPFGFILILVIFVTGLLLRKPNTYPKMIPEFFCDT SEQ ID NO: 62 LPFGFIL IL13Ralpha2epitope SEQ ID NO: 63 LFRAAQLANDVVLQIMEHLELASMTNMELMSSI Mad12VVISASIIVFNLLELEG SEQ ID NO: 64 LVQFIKDRISVVQA gp70CD4 peptide SEQ IDNO: 65 SPSYVYHQF gp70CD8 peptide SEQ ID NO: 66 ASMTNMELM adpgk peptideSEQ ID NO: 67 ATKNWPFL survivin20-28 SEQ ID NO: 68 TVSEFLKLsurvivin97-104 SEQ ID NO: 69 KRYKNRVASRKSRAKFKQLLQHYREVAAAKSSENZ13Mad12Anaxa DRLRLLLKLFRAAQLANDVVLQIMEHLELASMTNMELMSSIVVISASIIVFNLLELEGSTVHEILCKLSLEG DHSTPPSAYGSVKPYTNFDAE SEQ ID NO:70 MELAALCRWGLLLALLPPGAASTQVCTGTDMKL Her2/neuRLPASPETHLDMLRHLYQGCQVVQGNLELTYLP TNASLSFLQDIQEVQGYVLIAHNQVRQVPLQRLRIVRGTQLFEDNYALAVLDNGDPLNNTTPVTGA SPGGLRELQLRSLTEILKGGVLIQRNPQLCYQDTILWKDIFHKNNQLALTLIDTNRSRACHPCSPMCK GSRCWGESSEDCQSLTRTVCAGGCARCKGPLPTDCCHEQCAAGCTGPKHSDCLACLHFNHSGICE LHCPALVTYNTDTFESMPNPEGRYTFGASCVTACPYNYLSTDVGSCTLVCPLHNQEVTAEDGTQRC EKCSKPCARVCYGLGMEHLREVRAVTSANIQEFAGCKKIFGSLAFLPESFDGDPASNTAPLQPEQLQV FETLEEITGYLYISAWPDSLPDLSVFQNLQVIRGRILHNGAYSLTLQGLGISWLGLRSLRELGSGLALIH HNTHLCFVHTVPWDQLFRNPHQALLHTANRPEDECVGEGLACHQLCARGHCWGPGPTQCVNCS QFLRGQECVEECRVLQGLPREYVNARHCLPCHPECQPQNGSVTCFGPEADQCVACAHYKDPPFCV ARCPSGVKPDLSYMPIWKFPDEEGACQPCPINCTHSCVDLDDKGCPAEQRASPLTSIISAVVGILLVV VLGVVFGILIKRRQQKIRKYTMRRLLQETELVEPLTPSGAMPNQAQMRILKETELRKVKVLGSGAFGT VYKGIWIPDGENVKIPVAIKVLRENTSPKANKEILDEAYVMAGVGSPYVSRLLGICLTSTVQLVTQLM PYGCLLDHVRENRGRLGSQDLLNWCMQIAKGMSYLEDVRLVHRDLAARNVLVKSPNHVKITDFG LARLLDIDETEYHADGGKVPIKWMALESILRRRFTHQSDVWSYGVTVWELMTFGAKPYDGIPAREIP DLLEKGERLPQPPICTIDVYMIMVKCWMIDSECRPRFRELVSEFSRMARDPQRFVVIQNEDLGPASPL DSTFYRSLLEDDDMGDLVDAEEYLVPQQGFFCPDPAPGAGGMVHHRHRSSSTRSGGGDLTLGLEP SEEEAPRSPLAPSEGAGSDVFDGDLGMGAAKGLQSLPTHDPSPLQRYSEDPTVPLPSETDGYVAPLT CSPQPEYVNQPDVRPQPPSPREGPLPAARPAGATLERPKTLSPGKNGVVKDVFAFGGAVENPEYLTP QGGAAPQPHPPPAFSPAFDNLYYWDQDPPERGAPPSTFKGTPTAENPEYLGLDVPV SEQ ID NO: 71 LEEKKGNYVVTDHC EGFRvIII epitopeSEQ ID NO: 72 MELQAARACFALLWGCALAAAAAAQGKEVVLL EphA2DFAAAGGELGWLTHPYGKGWDLMQNIMND MPIYMYSVCNVMSGDQDNWLRTNWVYRGEAERIFIELKFTVRDCNSFPGGASSCKETFNLYYAESD LDYGTNFQKRLFTKIDTIAPDEITVSSDFEARHVKLNVEERSVGPLTRKGFYLAFQDIGACVALLSVRV YYKKCPELLQGLAHFPETIAGSDAPSLATVAGTCVDHAVVPPGGEEPRMHCAVDGEWLVPIGQCL CQAGYEKVEDACQACSPGFFKFEASESPCLECPEHTLPSPEGATSCECEEGFFRAPQDPASMPCTRPPS APHYLTAVGMGAKVELRWTPPQDSGGREDIVYSVTCEQCWPESGECGPCEASVRYSEPPHGLTRTS VTVSDLEPHMNYTFTVEARNGVSGLVTSRSFRTASVSINQTEPPKVRLEGRSTTSLSVSWSIPPPQQSR VWKYEVTYRKKGDSNSYNVRRTEGFSVTLDDLAPDTTYLVQVQALTQEGQGAGSKVHEFQTLSPE GSGNLAVIGGVAVGVVLLLVLAGVGFFIHRRRKNQRARQSPEDVYFSKSEQLKPLKTYVDPHTYEDP NQAVLKFTTEIHPSCVTRQKVIGAGEFGEVYKGMLKTSSGKKEVPVAIKTLKAGYTEKQRVDFLGEA GIMGQFSHHNIIRLEGVISKYKPMMIITEYMENGALDKFLREKDGEFSVLQLVGMLRGIAAGMKYLA NMNYVHRDLAARNILVNSNLVCKVSDFGLSRVLEDDPEATYTTSGGKIPIRWTAPEAISYRKFTSASD VWSFGIVMWEVMTYGERPYWELSNHEVMKAINDGFRLPTPMDCPSAIYQLMMQCWQQERARRP KFADIVSILDKLIRAPDSLKTLADFDPRVSIRLPSTSGSEGVPFRTVSEWLESIKMQQYTEHFMAAGYTAI EKVVQMTNDDIKRIGVRLPGHQKRIAYSLLGLKDQVNTVGIPI SEQ ID NO: 73 MDLVLKRCLLHLAVIGALLAVGATKVPRNQDW Gp100LGVSRQLRTKAWNRQLYPEWTEAQRLDCWRG GQVSLKVSNDGPTLIGANASFSIALNFPGSQKVLPDGQVIWVNNTIINGSQVWGGQPVYPQETDD ACIFPDGGPCPSGSWSQKRSFVYVWKTWGQYWQVLGGPVSGLSIGTGRAMLGTHTMEVTVYHR RGSRSYVPLAHSSSAFTITDQVPFSVSVSQLRALDGGNKHFLRNQPLTFALQLHDPSGYLAEADLSYT WDFGDSSGTLISRALVVTHTYLEPGPVTAQVVLQAAIPLTSCGSSPVPGTTDGHRPTAEAPNTTAG QVPTTEVVGTTPGQAPTAEPSGTTSVQVPTTEVISTAPVQMPTAESTGMTPEKVPVSEVMGTTLAEM STPEATGMTPAEVSIVVLSGTTAAQVTTTEWVETTARELPIPEPEGPDASSIMSTESITGSLGPLLDGTATLRLVKRQVPLDCVLYRYGSFSVTLDIVQGIESAEIL QAVPSGEGDAFELTVSCQGGLPKEACMEISSPGCQPPAQRLCQPVLPSPACQLVLHQILKGGSGTY CLNVSLADTNSLAVVSTQLIMPGQEAGLGQVPLIVGILLVLMAVVLASLIYRRRLMKQDFSVPQLPHS SSHWLRLPRIFCSCPIGENSPLLSGQQV SEQ IDNO: 74 MPRAPRCRAVRSLLRSHYREVLPLATFVRRLGPQ hTertGWRLVQRGDPAAFRALVAQCLVCVPWDARPP PAAPSFRQVSCLKELVARVLQRLCERGAKNVLAFGFALLDGARGGPPEAFTTSVRSYLPNTVTDALRG SGAWGLLLRRVGDDVLVHLLARCALFVLVAPSCAYQVCGPPLYQLGAATQARPPPHASGPRRRLG CERAWNHSVREAGVPLGLPAPGARRRGGSASRSLPLPKRPRRGAAPEPERTPVGQGSWAHPGRTRG PSDRGFCVVSPARPAEEATSLEGALSGTRHSHPSVGRQHHAGPPSTSRPPRPWDTPCPPVYAETKHF LYSSGDKEQLRPSFLLSSLRPSLTGARRLVETIFLGSRPWMPGTPRRLPRLPQRYWQMRPLFLELLGNH AQCPYGVLLKTHCPLRAAVTPAAGVCAREKPQGSVAAPEEEDTDPRRLVQLLRQHSSPWQVYGFV RACLRRLVPPGLWGSRHNERRFLRNTKKFISLGKHAKLSLQELTWKMSVRDCAWLRRSPGVGCVPA AEHRLREEILAKFLHWLMSVYVVELLRSFFYVTETTFQKNRLFFYRKSVWSKLQSIGIRQHLKRVQLREL SEAEVRQHREARPALLTSRLRFIPKPDGLRPIVNMDYVVGARTFRREKRAERLTSRVKALFSVLNYERAR RPGLLGASVLGLDDIHRAWRTFVLRVRAQDPPPELYFVKVDVTGAYDTIPQDRLTEVIASIIKPQNTY CVRRYAVVQKAAHGHVRKAFKSHVSTLTDLQPYMRQFVAHLQETSPLRDAVVIEQSSSLNEASSGL FDVFLRFMCHHAVRIRGKSYVQCQGIPQGSILSTLLCSLCYGDMENKLFAGIRRDGLLLRLVDDFLLV TPHLTHAKTFLRTLVRGVPEYGCVVNLRKTVVNFPVEDEALGGTAFVQMPAHGLFPWCGLLLDTRTL EVQSDYSSYARTSIRASLTFNRGFKAGRNMRRKLFGVLRLKCHSLFLDLQVNSLQTVCTNIYKILLLQA YRFHACVLQLPFHQQVWKNPTFFLRVISDTASLCYSILKAKNAGMSLGAKGAAGPLPSEAVQWLC HQAFLLKLTRHRVTYVPLLGSLRTAQTQLSRKLPGTTLTALEAAANPALPSDFKTILD SEQ ID NO: 75 MSPLWWGFLLSCLGCKILPGAQGQFPRVCMTVTRP-2 DSLVNKECCPRLGAESANVCGSQQGRGQCTEV RADTRPWSGPYILRNQDDRELWPRKFFHRTCKCTGNFAGYNCGDCKFGWTGPNCERKKPPVIRQ NIHSLSPQEREQFLGALDLAKKRVHPDYVITTQHWVGLLGPNGTQPQFANCSVYDFFVWLHYYSV RDTLLGGFFPWLKVYYYRFVIGLRVWQWEVISCKLIKRATTRQP SEQ ID NO: 76 MGVKASQTGFVVLVLLQCCSAYKLVCYYTSWS YKL-40QYREGDGSCFPDALDRFLCTHIIYSFANISNDHI DTWEWNDVTLYGMLNTLKNRNPNLKTLLSVGGWNFGSQRFSKIASNTQSRRTFIKSVPPFLRTHG FDGLDLAWLYPGRRDKQHFTTLIKEMKAEFIKEAQPGKKQLLLSAALSAGKVTIDSSYDIAKISQHLDF ISIMTYDFHGAWRGTTGHHSPLFRGQEDASPDRFSNTDYAVGYMLRLGAPASKLVMGIPTFGRSFTL ASSETGVGAPISGPGIPGRFTKEAGTLAYYEICDFLRGATVHRILGQQVPYATKGNQWVGYDDQESV KSKVQYLKDRQLAGAMVWALDLDDFQGSFCGQDLRFPLTNAIKDALAAT SEQ ID NO: 77 MAQLFLPLLAALVLAQAPAALADVLEGDSSEDRbrevican AFRVRIAGDAPLQGVLGGALTIPCHVHYLRPPPSRRAVLGSPRVKWTFLSRGREAEVLVARGVRVKV NEAYRFRVALPAYPASLTDVSLALSELRPNDSGIYRCEVQHGIDDSSDAVEVKVKGVVFLYREGSARY AFSFSGAQEACARIGAHIATPEQLYAAYLGGYEQCDAGWLSDQTVRYPIQTPREACYGDMDGFPG VRNYGVVDPDDLYDVYCYAEDLNGELFLGDPPEKLTLEEARAYCQERGAEIATTGQLYAAWDGGL DHCSPGWLADGSVRYPIVTPSQRCGGGLPGVKTLFLFPNQTGFPNKHSRFNVYCFRDSAQPSAIPE ASNPASNPASDGLEAIVTVTETLEELQLPQEATESESRGAIYSIPIMEDGGGGSSTPEDPAEAPRTLLEFETQSMVPPTGFSEEEGKALEEEEKYEDEEEKEEEEEEEEVEDEALWAWPSELSSPGPEASLPTEPAAQEESLSQ APARAVLQPGASPLPDGESEASRPPRVHGPPTETLPTPRERNLASPSPSTLVEAREVGEATGGPELSGV PRGESEETGSSEGAPSLLPATRAPEGTRELEAPSEDNSGRTAPAGTSVQAQPVLPTDSASRGGVAVVP ASGDCVPSPCHNGGTCLEEEEGVRCLCLPGYGGDLCDVGLRFCNPGWDAFQGACYKHFSTRRSW EEAETQCRMYGAHLASISTPEEQDFINNRYREYQWIGLNDRTIEGDFLWSDGVPLLYENWNPGQPD SYFLSGENCVVMVWHDQGQWSDVPCNYHLSYTCKMGLVSCGPPPELPLAQVFGRPRLRYEVDTV LRYRCREGLAQRNLPLIRCQENGRWEAPQISCVPRRPARALHPEEDPEGRQGRLLGRWKALLIPPSSP MPGP SEQ ID NO: 78MSRPQGLLWLPLLFTPVCVMLNSNVLLWLTALAI Neuroligin 4KFTLIDSQAQYPVVNTNYGKIRGLRTPLPNEILGP VEQYLGVPYASPPTGERRFQPPEPPSSWTGIRNTTQFAAVCPQHLDERSLLHDMLPIWFTANLDTLM TYVQDQNEDCLYLNIYVPTEDDIHDQNSKKPVMVYIHGGSYMEGTGNMIDGSILASYGNVIVITIN YRLGILGFLSTGDQAAKGNYGLLDQIQALRWIEENVGAFGGDPKRVTIFGSGAGASCVSLLTLSHYS EGLFQKAIIQSGTALSSWAVNYQPAKYTRILADKVGCNMLDTTDMVECLRNKNYKELIQQTITPATY HIAFGPVIDGDVIPDDPQILMEQGEFLNYDIMLGVNQGEGLKFVDGIVDNEDGVTPNDFDFSVSN FVDNLYGYPEGKDTLRETIKFMYTDWADKENPETRRKTLVALFTDHQWVAPAVATADLHAQYGSP TYFYAFYHHCQSEMKPSWADSAHGDEVPYVFGIPMIGPTELFSCNFSKNDVMLSAVVMTYWTNFA KTGDPNQPVPQDTKFIHTKPNRFEEVAWSKYNPKDQLYLHIGLKPRVRDHYRATKVAFWLELVPHL HNLNEIFQYVSTTTKVPPPDMTSFPYGTRRSPAKIWPTTKRPAITPANNPKHSKDPHKTGPEDTTVLIE TKRDYSTELSVTIAVGASLLFLNILAFAALYYKKDKRRHETHRRPSPQRNTTNDIAHIQNEEIMSLQMK QLEHDHECESLQAHDTLRLTCPPDYTLTLRRSPDDIPLMTPNTITMIPNTLTGMQPLHTFNTFSGGQ NSTNLPHGHSTTRV SEQ ID NO: 79MRILKRFLACIQLLCVCRLDWANGYYRQQRKLV PTPRz1EEIGWSYTGALNQKNWGKKYPTCNSPKQSPINI DEDLTQVNVNLKKLKFQGWDKTSLENTFIHNTGKTVEINLTNDYRVSGGVSEMVFKASKITFHWG KCNMSSDGSEHSLEGQKFPLEMQIYCFDADRFSSFEEAVKGKGKLRALSILFEVGTEENLDFKAIIDGV ESVSRFGKQAALDPFILLNLLPNSTDKYYIYNGSLTSPPCTDTVDWIVFKDTVSISESQLAVFCEVLTM QQSGYVMLMDYLQNNFREQQYKFSRQVFSSYTGKEEIHEAVCSSEPENVQADPENYTSLLVTWERP RVVYDTMIEKFAVLYQQLDGEDQTKHEFLTDGYQDLGAILNNLLPNMSYVLQIVAICTNGLYGKYS DQLIVDMPTDNPELDLFPELIGTEEIIKEEEEGKDIEEGAIVNPGRDSATNQIRKKEPQISTTTHYNRIGT KYNEAKTNRSPTRGSEFSGKGDVPNTSLNSTSQPVTKLATEKDISLTSQTVTELPPHTVEGTSASLNDGSKTVLRSPHMNLSGTAESLNTVSITEYEEESLLTSFKLDTGAEDSSGSSPATSAIPFISENISQGYIFSSENPETITYDVLIPESARNASEDSTSSGSEESLKDPSMEGN VWFPSSTDITAQPDVGSGRESFLQTNYTEIRVDESEKTTKSFSAGPVMSQGPSVTDLEMPHYSTFAYF PTEVTPHAFTPSSRQQDLVSTVNVVYSQTTQPVYNGETPLQPSYSSEVFPLVTPLLLDNQILNTTPAASSSDSALHATPVFPSVDVSFESILSSYDGAPLLPFSSASFSSELFRHLHTVSQILPQVTSATESDKVPLHASL PVAGGDLLLEPSLAQYSDVLSTTHAASETLEFGSESGVLYKTLMFSQVEPPSSDAMMHARSSGPEPSY ALSDNEGSQHIFTVSYSSAIPVHDSVGVTYQGSLFSGPSHIPIPKSSLITPTASLLQPTHALSGDGEWSGASSDSEFLLPDTDGLTALNISSPVSVAEFTYTTSVF GDDNKALSKSEIIYGNETELQIPSFNEMVYPSESTVMPNMYDNVNKLNASLQETSVSISSTKGMFPGS LAHTTTKVFDHEISQVPENNFSVQPTHTVSQASGDTSLKPVLSANSEPASSDPASSEMLSPSTQLLFYETSASFSTEVLLQPSFQASDVDTLLKTVLPAVPSDPI LVETPKVDKISSTMLHLIVSNSASSENMLHSTSVPVFDVSPTSHMHSASLQGLTISYASEKYEPVLLKSES SHQVVPSLYSNDELFQTANLEINQAHPPKGRHVFATPVLSIDEPLNTLINKLIHSDEILTSTKSSVTGKV FAGIPTVASDTFVSTDHSVPIGNGHVAITAVSPHRDGSVTSTKLLFPSKATSELSHSAKSDAGLVGGG EDGDTDDDGDDDDDDRGSDGLSIHKCMSCSSYRESQEKVMNDSDTHENSLMDQNNPISYSLSEN SEEDNRVTSVSSDSQTGMDRSPGKSPSANGLSQKHNDGKEENDIQTGSALLPLSPESKAWAVLTSDE ESGSGQGTSDSLNENETSTDFSFADTNEKDADGILAAGDSEITPGFPQSPTSSVTSENSEVFHVSEAEASNSSHESRIGLAEGLESEKKAVIPLVIVSALTFICLVVLVGILIYWRKCFQTAHFYLEDSTSPRVISTPPTPIFPISDDVGAIPIKHFPKHVADLHASSGFTEEFETLKEF YQEVQSCTVDLGITADSSNHPDNKHKNRYINIVAYDHSRVKLAQLAEKDGKLTDYINANYVDGYN RPKAYIAAQGPLKSTAEDFWRMIWEHNVEVIVMITNLVEKGRRKCDQYWPADGSEEYGNFLVTQKS VQVLAYYTVRNFTLRNTKIKKGSQKGRPSGRVVTQYHYTQWPDMGVPEYSLPVLTFVRKAAYAKR HAVGPVVVHCSAGVGRTGTYIVLDSMLQQIQHEGTVNIFGFLKHIRSQRNYLVQTEEQYVFIHDTL VEAILSKETEVLDSHIHAYVNALLIPGPAGKTKLEKQFQLLSQSNIQQSDYSAALKQCNREKNRTSSIIP VERSRVGISSLSGEGTDYINASYMGYYQSNEFIITQHPLLHTIKDFWRMIWDHNAQLVVMIPDGQN MAEDEFVYWPNKDEPINCESFKVTLMAEEHKCLSNEEKLIIQDFILEATQDDYVLEVRHFQCPKWPNP DSPISKTFELISVIKEEAANRDGPMIVHDEHGGVTAGTFCALTTLMHQLEKENSVDVYQVAKMINLM RPGVFADIEQYQFLYKVILSLVSTRQEENPSTSLDSNGAALPDGNIAESLESLV

1. A complex comprising: a) a cell penetrating peptide; b) at least oneantigen or antigenic epitope; and c) at least one TLR peptide agonist,wherein the components a)-c) are covalently linked.
 2. The complexaccording to claim 1, wherein the complex is a recombinant polypeptideor a recombinant protein.
 3. The complex according to claim 1, whereinthe cell penetrating peptide (i) has a length of the amino acid sequenceof said peptide of 5 to 50 amino acids in total, preferably of 10 to 45amino acids in total, more preferably of 15 to 45 amino acids in total;and/or (ii) has an amino acid sequence comprising a fragment of theminimal domain of ZEBRA, said minimal domain extending from residue 170to residue 220 of the ZEBRA amino acid sequence according to SEQ ID NO:3, wherein, optionally, 1, 2, 3, 4, or 5 amino acids have beensubstituted, deleted, and/or added without abrogating said peptide'scell penetrating ability, or a variant thereof. 4.-6. (canceled)
 7. Thecomplex according to claim 3, wherein the cell penetrating peptide hasan amino acid sequence comprising or consisting of an amino acidsequence according to SEQ ID NO: 6 (CPP3/Z13), SEQ ID NO: 7 (CPP4/Z14),SEQ ID NO: 8 (CPP5/Z15), or SEQ ID NO: 11 (CPPB/Z18), or sequencevariants thereof without abrogating said peptide's cell penetratingability, in particular sequence variants thereof sharing at least 70%sequence identity, preferably at least 80% sequence identity and morepreferably at least 90% sequence identity without abrogating saidpeptide's cell penetrating ability.
 8. The complex according to claim 1,wherein the at least one antigen or antigenic epitope is selected fromthe group consisting of: (i) a peptide, a polypeptide, or a protein,(ii) a polysaccharide, (iii) a lipid, (iv) a lipoprotein, (v) aglycolipid, (vi) a nucleic acid, and (vii) a small molecule drug or atoxin.
 9. The complex according to claim 1, wherein the at least oneantigen or antigenic epitope comprises or consists of at least onepathogen epitope and/or at least one tumor epitope, preferably the atleast one antigen or antigenic epitope comprises or consists of at leastone tumor epitope.
 10. (canceled)
 11. (canceled)
 12. The complexaccording to claim 1, wherein the complex comprises more than oneantigen or antigenic epitope, in particular 2, 3, 4, 5, 6, 7, 8, 9, 10or more antigens or antigenic epitopes, wherein the more than oneantigen or antigenic epitope, in particular 2, 3, 4, 5, 6, 7, 8, 9, 10or more antigens or epitopes, are positioned consecutively in thecomplex.
 13. (canceled)
 14. The complex according to claim 1, whereinthe at least one TLR peptide agonist is a TLR2, TLR4 and/or TLR5 peptideagonist, preferably a TLR2 peptide agonist and/or a TLR4 peptideagonist.
 15. The complex according to claim 14, wherein the at least oneTLR peptide agonist comprises or consists of an amino acid sequenceaccording to SEQ ID NO: 15 or a sequence variant thereof, in particulara sequence variant thereof sharing at least 70% sequence identity,preferably at least 80% sequence identity and more preferably at least90% sequence identity without abrogating said peptide's TLR agonistability. 16.-18. (canceled)
 19. The complex according to claim 2,wherein the components a) to c) are positioned in N-terminal→C-terminaldirection of the main chain of said complex in the order: (α) componenta)—component b)—component c); or (β) component c)—component a)—componentb), wherein the components may be linked by a further component, inparticular by a linker or a spacer.
 20. A nucleic acid encoding thecomplex according to claim 1, wherein the complex is a polypeptide or aprotein.
 21. A vector comprising the nucleic acid according to claim 20.22. A host cell comprising the vector according to claim
 21. 23.-24.(canceled)
 25. A cell loaded with a complex according to claim 1,wherein said cell is an antigen presenting cell, preferably a dendriticcell. 26.-27. (canceled)
 28. A vaccine comprising at least one of: (i) acomplex according to claim 1; (ii) a nucleic acid encoding the complexof (i); (iii) a vector comprising the nucleic acid according to (ii);(iv) a host cell comprising the vector according to (iii); or (v) a cellloaded with a complex according to (i).
 29. A pharmaceutical compositioncomprising at least one complex according to claim 1 and apharmaceutically acceptable carrier. 30.-40. (canceled)
 41. A method fortreating cancer or initiating, enhancing or prolonging ananti-tumor-response in a subject in need thereof comprisingadministering to the subject a complex comprising: a) a cell penetratingpeptide; b) at least one antigen or antigenic epitope; and c) at leastone TLR peptide agonist, wherein the components a)-c) are covalentlylinked.
 42. The method of claim 41, wherein the method comprisesadministering to the subject a cell loaded with the complex, apharmaceutical composition comprising the complex; and/or a vaccinecomprising i) the complex, ii) a nucleic acid encoding the complex, iii)a vector comprising the nucleic acid according to (ii), iv) a host cellcomprising the vector according to (iii), or v)a cell loaded with thecomplex.
 43. (canceled)
 44. A method for treating cancer and/or ahematological disorder, preferably a malignant neoplasm of the brain ora malignant neoplasm of lymphoid, hematopoietic and related tissue in asubject in need thereof, most preferably glioblastoma, comprisingadministering to the subject a complex comprising: a) a cell penetratingpeptide; b) at least one antigen or antigenic epitope; and c) at leastone TLR peptide agonist, wherein the components a)-c) are covalentlylinked.
 45. The method of claim 44, wherein the method comprisesadministering to the subject a cell loaded with the complex, apharmaceutical composition comprising the complex; and/or a vaccinecomprising i) the complex, ii) a nucleic acid encoding the complex, iii)a vector comprising the nucleic acid according to (ii), iv) a host cellcomprising the vector according to (iii), or v) a cell loaded with thecomplex.